Coordinating gene expression using RNA destabilizing elements

ABSTRACT

Control Devices are disclosed including RNA destabilizing elements (RDE), and RNA control devices, combined with transgenes, including Chimeric Antigen Receptors (CARs) in eukaryotic cells. RDEs can be combined with RNA control devices to make RDEs that include ligand mediated control. These smart RDEs and other RDEs can be used to optimize expression of transgenes, e.g., CARs, in the eukaryotic cells so that, for example, effector function is optimized. CARs and transgene payloads can also be engineered into eukaryotic cells so that the transgene payload is expressed and delivered at desired times from the eukaryotic cell.

This application is continuation of U.S. application Ser. No. 16/272,679filed Feb. 11, 2019, which claims priority to U.S. provisionalapplication Ser. No. 62/630,191 filed Feb. 13, 2018.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CBIO030_ST25.txt”, a creation date of Feb. 11, 2018, and asize of 9 kilobytes. The Sequence Listing filed via EFS-Web is part ofthe specification and is incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

Chimeric Antigen Receptors are human engineered receptors that maydirect a T-cell to attack a target recognized by the CAR. For example,CAR T cell therapy has been shown to be effective at inducing completeresponses against acute lymphoblastic leukemia and other B-cell-relatedmalignancies and has been shown to be effective at achieving andsustaining remissions for refractory/relapsed acute lymphoblasticleukemia (Maude et al., NEJM, 371:1507, 2014). However, dangerous sideeffects related to cytokine release syndrome (CRS), tumor lysis syndrome(TLS), B-cell aplasia and on-tumor, off-target toxicities have been seenin some patients.

There are currently two extant strategies to control CAR technology. Thefirst is an inducible “kill switch.” In this approach, one or more“suicide” genes that initiate apoptotic pathways are incorporated intothe CAR construct (Budde et al. PLoS1, 2013doi:10.1371/journal.pone.0082742). Activation of these suicide genes isinitiated by the addition of AP1903 (also known as rimiducid), alipid-permeable tachrolimus analog that initiates homodimerization ofthe human protein FKBP12 (Fv), to which the apoptosis-inducing proteinsare translationally fused. In the ideal scenario, these kill switchesendeavor to sacrifice the long-term surveillance benefit of CARtechnology to safeguard against toxicity. However, in vivo, thesesuicide switches are not likely to realize this goal, as they areoperating against powerful selection pressures for CAR T-cells that donot respond to AP1903, a situation worsened by the inimical error-proneretroviral copying associated with the insertion of stable transgenesinto patient T-cells. In this scenario, non-responsive CAR T-cell cloneswill continue to proliferate and kill target cells in anantigen-dependent manner. Thus, kill switch technology is unlikely toprovide an adequate safeguard against toxicity.

The second CAR regulatory approach is transient CAR expression, whichcan be achieved in several ways. In one approach, T-cells are harvestedfrom unrelated donors, the HLA genes are deleted by genome-editingtechnology and CAR-encoding transgenes are inserted into the genome ofthese cells. Upon adoptive transfer, these CAR T-cells will berecognized by the recipient's immune system as being foreign anddestroyed, thus the CAR exposure in this system is transient. In anothertransient CAR exposure approach, mRNA of a CAR-encoding gene isintroduced into harvested patient T-cells (Beatty, G L 2014. CancerImmunology Research 2 (2): 112-20. doi:10.1158/2326-6066.CIR-13-0170).As mRNA has a short half-life and is not replicated in the cell orstably maintained, there is no permanent alteration of theCAR-expressing T-cell, thus the CAR expression and activity will be fora short period of time. However, as with the kill-switch approach, thesetransient CAR exposure approaches sacrifice the surveillance benefit ofCARs. Additionally, with these transient systems acute toxicity can bedifficult to control.

SUMMARY OF THE INVENTION

In an aspect, the description discloses a eukaryotic cell with a CAR,T-cell receptor, or other targeting polypeptide and a transgene underthe control of an RNA Destabilizing Element (RDE). The RDE may controlmultiple transgenes or multiple RDEs may control multiple transgenes.The multiple transgenes may be arranged serially and/or as a concatemerand/or in other arrangements. Multiple RDEs may be used to regulate atransgene, and these multiple RDEs can be organized as a concatemer,interspersed within a region of the transcript, or located in differentparts of the transcript. Multiple transgenes can be regulated by an RDEor a combination of RDEs. The RDEs can be localized in the 3′-UTR, the5′-UTR and/or an intron. RDEs can include, for example, the RDEs from AU1 (CD40L), AU 2 (CSF2), AU 3 (CD247), AU 4 (CTLA4), AU 5 (EDN1), AU 6(IL2RA), AU 7 (SLC2A1), AU 8 (TRAC), AU 9 (CD274), AU 10 (Myc), AU 11(CD19), AU 12 (IL4), AU 13 (IL5), AU 14 (IL6), AU 15 (IL9), AU 16(IL10), AU 17 (IL13), AU 18 (FOXP3), AU 19 (TMEM-219), AU 20(TMEM-219snp), AU 21 (CCR7), AU 22 (SEM-A4D), AU 23 (CDC42-SE2), AU 24(CD8), AU 27 (bGH), and/or AU 101 (Interferon gamma or IFNg). Other RDEsare disclosed in the following description.

In an aspect, the RDE can be under the control of a RNA control device.Such, Smart RDEs place the RDE control under the regulation of the RNAcontrol device which introduces ligand control to the RDE. The RNAcontrol device can disrupt the RDE when ligand is bound (or not bound)resulting in loss of the RDE control, and when ligand is added (orremoved) the RNA control device is inhibited and the RDE structure isavailable for interaction with RNA binding proteins. The RNA controldevice could also act upon a portion of the transcript that disrupts theRDE (e.g., the portion of the transcript could form secondary structureswith the RDE that inhibit RNA binding proteins from binding to the RDE),when the RNA control device binds (or is free from ligand) the RNAcontrol device disrupts the inhibitory portion of the transcript so itis not available to interact with the RDE, and the RDE is now availableto interact with RNA binding proteins. This RNA control deviceregulation allows the activity of the RDE to be ligand controlledthrough the action of the RNA control device.

In an aspect, an RDE, combination of RDEs, and/or modified RDEs can beused to provide desired kinetic parameters to the regulation of a geneproduct including, for example, amount of expression, steady stateconcentration, C_(max) (maximal concentration of gene product obtained),T_(max) (time to reach C_(max)), baseline expression, speed of induction(acceleration), induction rate (velocity), dynamic range also known asfold regulation (induced expression/basal expression), maximal dynamicrange (DR_(max)), time to DR_(max), area under the curve (AUC), etc. ARDE construct can be made that has a desired set of kinetic parametersto provide the level, degree, temporal, and amount of regulation that isdesired. In addition, RDE concatemers can be used to alter the kineticperformance of a construct.

Combinations of RDEs can be used to provide temporal regulation betweentwo or more transgenes. RDEs can be selected to provide maximal rates ofexpression (and different amounts of maximal expression) at differenttimes following activation of a cell (or induction of expression). Thistemporal control allows a first transgene encoded polypeptide to alterthe state of the cell so that the cell is prepared to be acted upon by asecond polypeptide encoded by a second transgene with an RDE thatprovides later in time expression. This temporal control can also beused to time the expression of two, three or more transgenes followingactivation of a cell. If the transgene encoded polypeptides aresecreted, they can act in a temporal fashion upon target cells. Forexample, a first transgene polypeptide (with an early expression RDE)could be secreted and act upon a target cell to change its state (e.g.,induce the expression of receptor). The second transgene polypeptide isexpressed at a later time (under the control of a later expression RDE)and acts upon the target cell with the changed state (e.g., the secondprotein can be a ligand for the induced receptor).

In an aspect, the RDE can be engineered to increase or decrease thebinding affinity of RNA binding protein(s) that interact with the RDE.Altering the affinity of the RNA binding protein can change the timingand response of transgene expression as regulated by the RNA bindingprotein. In an aspect, the RNA binding protein binding at the RDE isaltered by the metabolic state of the cell and changing the bindingaffinity of the RDE for the RNA binding protein alters the response toand/or timing of transgene expression with the metabolic state of thecell. In an aspect, the RNA binding protein binding at the RDE isaltered by the redox state of the cell and changing the binding affinityof the RDE for the RNA binding protein alters the response to and/ortiming of transgene expression with the redox state of the cell.

In an aspect, the CAR, T-cell receptor, B-cell receptor, innate immunityreceptor, or other targeting receptor or targeting polypeptiderecognizes an antigen at the target site (e.g., tumor cell or otherdiseased tissue/cell) and this activates the cell. The transgene can beanother CAR that recognizes a second antigen at the target site andactivation of the cell by the first CAR, T-cell receptor or othertargeting polypeptide induces the second CAR allowing the eukaryoticcell to recognize the target site by a second antigen. In an aspect, theeukaryotic cell has a first CAR that recognizes an antigen at a targetsite and this activates a transgene (through an RDE) that encodes apolypeptide that directly or indirectly reduces the activation state ofthe cell. For example, the transgene may encode a second CAR thatrecognizes an antigen on healthy tissue so that when the first CARreacts with antigen at a nontarget cell, the eukaryotic cell will bede-activated by the second CAR interaction with the healthy cell antigen(that is not present or is present in reduced amounts at the targetsite).

In some aspects, the eukaryotic cell is an immune cell, e.g., a T-cell,a natural killer cell, a B-cell, a macrophage, a dendritic cell, orother antigen presenting cell. In these aspects, activation of the cellby the CAR or changing the metabolic state of the immune cell in otherways can induce expression of the transgene through the RDE. The RDEthat controls the transgene can have microRNA binding sites and can beengineered to remove one or more of these microRNA binding sites. TheRDE can be bound by the Hu Protein R (HuR). Without wishing to be boundby theory it is expected that HuR can bind to some RDEs, and act tostabilize the mRNA, leading to enhanced translation. Some RDEs can betied to the glycolytic state of the eukaryotic cell through the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH), other dehydrogenases,other oxidoreductases, or other glycolytic enzymes that can bind to anRDE when the eukaryotic cell is not activated (low glycolytic activity),quiescent, or at rest. When GAPDH or the other enzymes bind to the RDEthis can reduce half-life of the RNA with the RDE. In this aspect, CARactivation of the eukaryotic cell (e.g., T-lymphocyte) can induceglycolysis in the cell which reduces GAPDH binding of the RNA, increaseshalf-life of the RNA, which produces increased expression of thetransgene encoded in the RNA and controlled by the RDE. Without wishingto be bound by theory, as GAPDH vacates the RDE, HuR or other RDEbinding proteins may subsequently bind either the same RDE, or apreviously inaccessible RDE (sterically hindered by presence of GAPDH),further stabilizing the mRNA, increasing half-life of the mRNA, andproducing further increased expression of the transgene encoded by theRNA and controlled by said RDE. Thus, CAR activation can induceexpression of the transgene. In other aspects, other activation of theimmune cell can cause GAPDH to engage in glycolysis and so induceexpression of the transgene under the control of the RDE.

Expression from the transcript with the RDE(s) can respond to themetabolic state of the cell. For example, the RDE can be bound bymetabolic or glycolytic enzymes which couples expression of thetransgene to the activation state of the cell through these metabolic orglycolytic enzymes. Some metabolic or glycolytic enzymes bind to RDEs inthe transcript and degrade or target for degradation the transcript.When those metabolic or glycolytic enzymes become active, the enzymes nolonger bind to the RDEs, the transcripts are stable for a longer periodof time, and the transcripts can be translated for this longer period oftime. Cells expressing transgenes under the control of such RDEs canalso be engineered to express a CAR that can alter the metabolic stateof the cell at desired times resulting in expression of the transgene atthe desired time. Alternatively, other stimuli can be used to alter themetabolic state of the eukaryotic cell resulting in expression of thetransgene. For example, the metabolic state of the cell can be alteredto cause transgene expression (or to inhibit expression) by stimuliincluding, for example, small molecules (e.g., PMA/ionomycin),cytokines, a TCR and costimulatory domain engagement with ligand, oxygenlevels, cellular stress, temperature, or light/radiation.

GAPDH binding to the RDE can be increased by introducing into the cell asmall molecule that inhibits glycolysis such as, for example,dimethylfumarate (DMF), rapamycin, 2-deoxyglucose, 3-bromophyruvic acid,iodoacetate, fluoride, oxamate, ploglitazone, dichloroacetic acid, orother metabolism inhibitors such as, for example,dehydroepiandrosterone. Other small molecules can be used to reduceGAPDH binding to the RDE. Such small molecules may block the RDE bindingsite of GAPDH including, for example, CGP 3466B maleate or Heptelidicacid (both sold by Santa Cruz Biotechnology, Inc.), pentalenolactone, or3-bromopyruvic acid. Other small molecules can be used to analogouslyinhibit other enzymes or polypeptides from binding to RDEs. Other smallmolecules can be used to change the redox state of GAPDH, leading to analtered affinity of GAPDH for the RDE. Other small molecules known tointeract with GAPDH function, such as vitamin C, saframycin, salicylicacid, insulin, vitamin d3, metformin, or suramin can modify the bindingof GAPDH for the RDE. Other molecules can modify the binding of GAPDHfor the RDE including trehalose, galactose and other saccharides. Othermolecules known to alter GAPDH structure can modify the binding of GAPDHfor the RDE including nitric oxide and hydrogen sulfide.

In an aspect, activation of the immune cell induces expression of thetransgene that can encode a payload to be delivered at the target(activation) site. The transgene can encode a payload for delivery atthe site of CAR activation and/or immune cell activation and/or otherreceptor activation. The payload can be a cytokine, an antibody, areporter (e.g., for imaging), a receptor (such as a CAR), or otherpolypeptide that can have a desired effect at the target site. Thepayload can remain in the cell, or on the cell surface to modify thebehavior of the cell. The payload can be an intracellular protein suchas a kinase, phosphatase, metabolic enzyme, an epigenetic modifyingenzyme, a gene editing enzyme, etc. The payload can be a gene regulatoryRNA, such as, for example, siRNA, microRNAs (e.g., miR155), shRNA,antisense RNA, ribozymes, and the like, or guide RNAs for use withCRISPR systems. The payload can be a nucleic acid (e.g., a vector, or ahuman artificial chromosome (HAC)). The payload can also be a membranebound protein such as GPCR, a transporter, etc. The payload can be animaging agent that allows a target site to be imaged (target site has adesired amount of target antigen bound by the CAR). The payload can be acheckpoint inhibitor, and the CAR and/or other binding protein (e.g.,T-cell receptor, antibody or innate immunity receptor) can recognize atumor associated antigen so the eukaryotic cell preferentially deliversthe checkpoint inhibitor at a tumor. The payload can be a cytotoxiccompound including, for example, a granzyme, an apoptosis inducer, acytotoxic small molecule, or complement. The payload can be an antibody,such as for example, an anti-4-1BB agonist antibody (an anti-CD137antibody), an anti-IL 1b antibody (anti-inflammatory),anti-CD29/anti-VEGF antibody, an anti-CTLA4 antibody, a bispecificantibody (e.g., BiTE), or an anti-CD11b antibody. The payload can be animmune polypeptide, including for example, cytokines (e.g., IL-2, IL-12,IL-15, IL-18), chemokines (e.g., CXCL12), perforins, granzymes, andother immune polypeptides. The payload can be an enzyme including forexample, hyaluronidase, or heparinase. The payload can be a polypeptideincluding for example, CCR2, CCR4, a BiTE (activates immunosuppressedT-cells), soluble CD40 ligand, HSP70, and HSP60. The payload can be atransgene(s) which delivers a virus as a payload. For example, the RDEcan control a master control element that controls the expression of thevirus genes for replication and coat/envelope proteins. Alternatively,the Rep and coat/envelope proteins can be placed under the control ofinducible promoters that are controlled by a regulatory protein, andthat regulatory protein can be controlled by an RDE. Stillalternatively, the Rep proteins of the virus can be placed under thecontrol of an RDE, and/or the coat/envelope proteins of the virus can beplaced under the control of an RDE. As with other payloads this complexpayload can use CAR T-cell regulation or any other regulation thatinduces glycolysis in a cell. Helper constructs in a T cell, or otherdelivery cell can encode the genes needed for viral replication andviral packaging.

Additional constructs can be employed that encode viral coat/envelopepolypeptides for the viral capsid and enzymes for viral replication (Repproteins), lysis of the host cell, etc. Packaging and other helperconstructs can include, but are not limited to, for example, lentiviralpackaging (helper) systems (e.g., available from Clontech/Takara andaddgene), pSV-A-MLV-env (NIH catalog number: 1065), which contains theamphotropic murine leukemia virus env gene linked to the MLV LTR and anSV origin, wherein the cloning vector is PSV7d; HlVgpt (NIH catalognumber: 1067), which is an Xbal-Hpat pHXB2gpt fragment (Drs. A. Fisherand F. Wong-Staal) containing pro-viral and flanking cellular sequencescloned into the HincII-Xbal sit of pBS KS (+/−); PsV-Ψ-MLV-env (NIHcatalog number: 3422), which is Ψ-Moloney Murine Leukemia virus DNA(from Richard Mann) cloned into the SV40 expression vector pSV7d at theEcoR1 site; and psPAX2 (equivalent to pCMV δR8.91), wherein the plasmidencodes for the Gag/Pro/Pol genes derived from HIV-1. The promoter isthe chicken beta actin promoter and polyadenylation signal is the rabbitbeta globin polyA.

Expression from helper constructs (e.g., Rep constructs and/orcoat/envelope protein constructs) or the virus (e.g., oncolytic viruses)is placed under the control of an RDE. The RDE can be one that isactivated for expression when a receptor activates the host cell (e.g.,when receptor binding can alter the energy state of the cell byactivating glycolysis or other energy pathways). For example, the Repand Cap genes of a virus (or helper construct) can be placed under thecontrol of an RDE that provides expression after receptor activation ofthe host cell (e.g., induction of glycolysis). Other virus genes such asthose encoding master switch polypeptides that control transcription,e.g., (for adenovirus) E1A, E1B, E2A, E4ORF6 and/or VARNA can also beplaced under the control of an RDE that provides expression afterreceptor activation of the host cell (or other systems as describedabove). Using RDEs with virus polypeptides in this manner ties virusproduction in the host cell to activation of the host cell through thereceptor (which binds a ligand at the target site).

A variety of constructs can be delivered as viral payload ranging fromfull virus to transfer constructs (contain packaging signals andtransgenes). Infective constructs can also encode desired transgenes andnoninfective constructs can encode polypeptides that kill infected cellsbut do not produce infective virus (e.g., the construct could includeviral functions that lyse the infected cell, or the construct couldencode viral proteins that are displayed on the infected cell forrecognition by cytotoxic T-cells or natural killer cells). Noninfectiveconstructs can also deliver transgenes to the target cell that alter thegenotype and/or phenotype of the target cell. For example, noninfectivetransfer constructs can deliver transgenes for gene therapy. Transferconstructs are available from addgene as transfer plasmids. Viralpayloads can contain polypeptides that serve as marker proteins toassess cell transformation and expression, fusion proteins, polypeptideshaving a desired biological activity, gene products that can complementa genetic defect, RNA molecules, transcription factors, other geneproducts that are of interest in regulation and/or expression, and otherpayloads described herein. The payload may also contain nucleotidesequences that provide a desired effect or regulatory function (e.g.,transposons, transcription factors).

The viral payload may be a transgene encoding a gene therapy product. Agene therapy product may include, but is not limited to, a polypeptide,RNA molecule, or other gene product that, when expressed in a targetcell, provides a desired effect. The gene therapy product may contain asubstitute for a non-functional gene that is absent or mutated in thetarget cell. The viral payload may be a transgene that will target thecell for killing by the immune system (e.g., cytotoxic T-cells and/ornatural killer cells). For example, the transgene could encode theheterologous polypeptide that is displayed on the surface of theinfected cell and that cytotoxic T-cells and/or natural killer cellsrecognize as targets to be killed. For example, the heterologouspolypeptide could be a heterologous MHC polypeptide that is incompatiblewith the host, or a viral polypeptide that will trigger an immuneresponse to destroy the infected cells (the immune system recognizes thecells as virus infected and kills the cells). The transgene could alsobe one of, for example, a Fas, a TNFαR, a DR3, a DR4, or a DR5polypeptide (which can act as apoptosis receptors).

A viral payload construct encoding a payload may contain or encode aselectable marker. A selectable marker may comprise a gene sequence or aprotein encoded by a gene sequence expressed in a host cell that allowsfor the identification, selection, and/or purification of the host cellfrom a population of cells that may or may not express the selectablemarker. The selectable marker provides resistance to survive a selectionprocess that would otherwise kill the host cell, such as treatment withan antibiotic. An antibiotic selectable marker may contain one or moreantibiotic resistance factors, including but not limited to neomycinresistance (e.g., neo), hygromycin resistance, kanamycin resistance,and/or puromycin resistance.

A payload construct encoding a payload may contain a selectable markerthat includes, but is not limited to, β-lactamase, luciferase,β-galactosidase, or a reporter gene as that term is understood in theart, including cell-surface markers, such as CD4 or the truncated nervegrowth factor (NGFR) (for GFP, see WO 96/23810; Heim et al., CurrentBiology, 2:178-182 (1996); Heim et al., Proc. Natl. Acad. Sci. USA,(1995); or Heim et al., Science, 373:663-664 (1995); for β-lactamase,see WO 96/30540); the contents of each of which are herein incorporatedby reference in their entirety.

A viral payload containing a nucleic acid for expression in a targetcell can be incorporated into the viral genome located between two ITRsequences, or on either side of an asymmetrical ITR engineered with twoD regions. A payload construct encoding one or more payloads forexpression in a target cell may contain one or more payload ornon-payload nucleotide sequences operably linked to at least one targetcell-compatible promoter. Such payload constructs can be made fromtransfer plasmids that are available from addgene. A person skilled inthe art will recognize that a target cell may require a specificpromoter including, but not limited to, a promoter that is speciesspecific, inducible, tissue-specific, or cell cycle-specific (Parr etal., Nat. Med. 3:1145-9 (1997).

The transgene or payload can be carried on any suitable vector, e.g., aplasmid, which is delivered to a host cell such as the transfer plasmidsavailable from addgene. Plasmids may be engineered such that they aresuitable for replication and, optionally, integration in prokaryoticcells, mammalian cells or both. These plasmids contain sequencespermitting replication of the transgene in eukaryotes and/or prokaryotesand selection markers for these systems. Selectable markers or reportergenes may include sequences encoding gentamycin, hygromicin or purimycinresistance, among others. The plasmids may also contain certainselectable reporters or marker genes that can be used to signal thepresence of the vector in bacterial cells, such as ampicillinresistance. Other components of the plasmid may include an origin ofreplication and an amplicon, such as the amplicon system employing theEpstein Barr virus nuclear antigen. This amplicon system, or othersimilar amplicon components permit high copy episomal replication in thecells.

The construct carrying the transgene or payload is transfected into thecell, where it may exist transiently. Alternatively, the transgene maybe stably integrated into the genome of the host cell, eitherchromosomally or as an episome. The transgene may be present in multiplecopies, optionally in head-to-head, head-to-tail, or tail-to-tailconcatamers.

In some aspects, the expression of CAR, DE-CAR and/or Side-CARpolypeptide is controlled, at least in part, by an RDE that interactswith a glycolytic enzyme with RDE binding activity, e.g., GAPDH. Theglycolytic enzyme can bind to the RDE and reduce production of the CAR,DE-CAR, Side-CAR polypeptide, and/or other transgene product. Thisreduction in polypeptide production can occur because of an inhibitionof translation and/or an increase in the rate of mRNA degradation (RDEbinding can shorten the half-life of the mRNA). Some RDE bindingproteins may reduce translation and enhance degradation of RNA to reducethe level of polypeptide made. The RDE can be an AU rich element fromthe 3′ UTR of a transcript (e.g., a transcript encoding IL-2 or IFN-γ),or can be a modified 3′ UTR that has been engineered to remove one ormore microRNA sites (e.g., modified 3′-UTRs of IL-2 or IFN-γ). In anaspect, the expression of the transgene, CAR, DE-CAR and/or Side-CARpolypeptide under the control of an RDE bound by a glycolytic enzyme(s),e.g., GAPDH, is increased by increasing the activity of the enzyme(s) inprosecuting glycolysis. The activity of enzymes in glycolysis can beincreased by providing the cell with increased glucose in the cellmedium, increasing triose isomerase activity in the cell, or providingthe cell with a compound that increases glycolysis in the cell, e.g.,tamoxifen or glucose. The RDE can bind to Hu Protein R (HuR). Withoutwishing to be bound by theory it is expected that HuR binds to someAU-rich RDEs and U-rich RDEs, and can act to stabilize the mRNA, leadingto enhanced translation. Thus, cell conditions that result in increasedHuR expression can increase expression of transgenes with appropriateAU-rich elements and/or U-rich elements, and conditions that reduce HuRexpression can decrease expression of these transgenes. HuR interactionwith the 3′ UTR of the transgene (or native genes) can also be alteredby expressing a recombinant transcript containing HuR binding sites.Expression of these transcripts will reduce the amount of HuR availableto bind to the transgene transcript or native HuR regulated transcriptsand reduce the half-lives of these transcripts resulting in decreasedexpression.

In an aspect, RDE control can be used to lower CAR expression in asubject which can reduce the availability of CAR polypeptide for immunereactions. This can lower the immunogenicity of transgenic immune cellswith the CAR. In part, this lower immunogenicity occurs because the CARpeptide has lower exposure to the immune system.

In an aspect, nucleic acids can be used to boost the response of immunecells upon stimulation of the immune cell. For example, the immune cellcan produce higher amounts of immune polypeptides (greater C_(max)) withfaster kinetics of production. The immune polypeptides can include, forexample, cytokines, perforins, granzymes, apoptosis inducingpolypeptides, etc. The nucleic acids that boost the immune response cancomprise control regions operably linked to nucleic acids encoding RDEsfor selected RDE binding proteins, so that upon expression of thenucleic acid into RNA the RDEs in the RNA bind the RDE binding proteinsthat repress expression of a polypeptide, for example, cytokines,perforins, granzymes, and other immune polypeptides. The expression ofthe RNAs with the RDEs can poise the eukaryotic cell for expression ofpolypeptide controlled by RDEs. For example, the expression of RNAs withthe RDEs may be done in immune cells to poise the cell for expression ofimmune polypeptides upon stimulation of the immune cell.

In an aspect, the CAR, DE-CAR, Side-CAR polypeptides, and/or otherreceptor can be directed against antigens found on acute myeloidleukemia (AML) cells including, for example, CD 33, CD 34, CD 38, CD43,CD 44, CD 45, CD 45RA, CD 47, CD 64, CD 66, CD 123, CD 133, CD 157,CLL-1, CXCR4, LeY, PR1, RHAMM (CD 168), TIM-3, and/or WT1. Themonoclonal antibody 293C3-SDIE can be used as the extracellular elementfor the CAR, DE-CAR and/or Side-CAR polypeptides. (Rothfelder et al.,2015, at ash.confex.com/ash/2015/webprogram/Paper81121.html, which isincorporated by reference in its entirety for all purposes) Otherantigens for AML are known in the art and may be the target of the CAR,DE-CAR, Side-CAR, and/or other receptor. An onco-sialylated CD 43 hasbeen associated with acute myeloid leukemia (AML) and thisonco-sialylated CD 43 is not found on normal cells and tissue. Thisonco-sialylated CD 43 is bound by the monoclonal antibody AT14-013, andthe variable region of this antibody is used to make an anti-oncosialylated CD 43 CAR. AT14-013 recognizes the unique sialylation epitopefound on this onco-sialylated CD 43. This CAR is specific for AML anddoes not have side reactivity with normal tissue in a subject. In anaspect, the CAR, DE-CAR, Side-CAR polypeptides, and/or other receptorcan be directed against antigens found on diffuse large cell B-celllymphoma (DLBCL) cells including, for example, CD19, CD20, CD22, CD79a,CD5, CD10, and CD43. Other antigens for DLBCL are known in the art andmay be the target of the CAR, DE-CAR, Side-CAR, and/or other receptor.

Other antigens that can be targeted by the CAR, DE-CAR, side-CAR orother receptor include, for example, DLL3, HER2, PSCA, CSPG4, EGFRvIII,MSLN (mesothelin), FAP, MUC16 (CA-125), CEA, CD133 (PROM1), IL13Ra,CD171 (L1CAM), CD123, (IL3R), CD33 (SIGLEC3), LeY, GUCY2C, BCMA and/orEPHA2. Eukaryotic cells with CAR, DE-CAR, side-CAR or other receptorstargeting these antigens can include a payload such as, for example, oneor more of anti-4-1BB antibody, anti-CD11b antibody, anti-CTLA4antibody, anti-ILlb antibody, a BiTE, CCL2, anti-CXCR4 antibody,anti-CXCL12 antibody, HAC, heparinase, hyaluronidase, Hsp60, Hsp70,IL-2, IL-12, IL-15, IL-18, INFγ, miRNA (e.g., mir155), and/or CD40ligand.

The CAR, DE-CAR, Side-CAR polypeptides, and/or other receptor can bedirected against antigens found on solid tumors such as, for example,integrins such as αvβ6 (found on numerous solid tumors (including, forexample, oral squamous cell cancer, colon cancer, pancreatic cancer,gastric cancer, breast cancer, ovarian cancer, cervical cancer, lungcancer, etc.).

In an aspect, small molecules and other molecules that affect theavailability of GAPDH or other RDE binding proteins to bind RDEs can beused to regulate gene expression by GAPDH, other RDE binding glycolysisenzymes, and/or other RDE binding enzymes involved in energy and cellmetabolism. Molecules that increase glycolysis in a cell can reduce theamount of GAPDH available for binding to RDEs which can increasetranslation from transcripts under GAPDH control. Similarly, otherglycolysis enzymes and metabolic enzymes can bind to RDEs and activatingglycolysis and other energy pathways in the cell can reduce the amountof these enzymes that are available to bind their corresponding RDEs.This reduced binding can increase translation from transcriptscontrolled by these RDE binding proteins (enzyme binding to the RDEdecreases expression) or can decrease translation if enzyme binding hasa positive effect on expression. These molecules can also be useful inthe treatment of certain types of neural degeneration associated withinflammation and/or autoimmune diseases. These molecules can be used toalter the amount of GAPDH in immune cells so that RDEs are bound and theimmune cells reduce expression of RDE regulated genes. Some genes underRDE control in immune cells are associated with inflammation and so,molecules that increase the amount of RDE binding proteins that inhibitthe inflammatory associated transcripts could reduce inflammation.

A nucleic acid construct encoding a transcript with selected RDEs can beexpressed in an immune cell, for example, a T-lymphocyte. Therecombinant transcript with the selected RDEs can bind to and depletethe levels of RDE binding proteins in the T-lymphocyte so thattranscripts encoding polypeptides regulated by the depleted RDE bindingproteins are expressed at different threshold points of activation forother cellular signals. The use of the RDE constructs can increase thekinetics of expression and/or the Cmax of expression of the polypeptideswhose expression is controlled by the RDE.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram for optimal CAR activity where the threevariables are CAR copy number, target epitope copy number and CARbinding affinity.

FIG. 2 shows a graph for the bioluminescence from T-cells withluciferase controlled by an RDE following activation of the T-cell byRaji target cells (activate CAR) or by CD3/CD28 beads (activate TCR) ascompared to bioluminescence of T-cells at resting.

FIG. 3 shows a graph for bioluminescence from T-cells with luciferasecontrolled by the RDEs Gold1, Gold2, or Gold3 following activation ofthe T-cell by Raji target cells (activate CAR) as compared tobioluminescence of T-cells at resting.

FIG. 4 shows a graph for the IL-12 expression from T-cells with IL-12expression controlled by an RDE following activation of the T-cell byRaji target cells (activate CAR) as compared to IL-12 expression ofT-cells at resting.

FIG. 5 shows basal luciferase expression and activated luciferaseexpression for luciferase constructs utilizing different RDEs as controlelements in Jurkat cells.

FIG. 6 shows basal luciferase expression and activated luciferaseexpression for luciferase constructs utilizing different RDEs as controlelements in primary T-cells.

FIG. 7 shows activated luciferase/basal luciferase expression after 1,3, 6, and 8 days for luciferase constructs utilizing different RDEs ascontrol elements.

FIG. 8 shows basal luciferase expression and activated luciferaseexpression for luciferase constructs utilizing different RDEs as controlelements.

FIG. 9 shows the dynamic range (activated luciferase/basal luciferase)measured 1, ¾, 6, and 8 days after activation for luciferase constructsutilizing different RDEs as control elements.

FIG. 10 shows the dynamic range (activated luciferase/basal luciferase)measured 1, ¾, 6, and 8 days after activation for luciferase constructsutilizing different RDEs as control elements.

FIG. 11 shows the impact on luciferase expression for luciferaseconstructs utilizing an RDE as a control element in the presence ofglucose and galactose.

DETAILED DESCRIPTION OF THE INVENTION

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimscan be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.Numerical limitations given with respect to concentrations or levels ofa substance are intended to be approximate, unless the context clearlydictates otherwise. Thus, where a concentration is indicated to be (forexample) 10 μg, is intended that the concentration be understood to beat least approximately or about 10 μg.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings.

As used herein, an “actuator element” is defined to be a domain thatencodes the system control function of the RNA control device. Theactuator domain can optionally encode the gene-regulatory function.

As used herein, an “antibody” is defined to be a protein functionallydefined as a ligand-binding protein and structurally defined ascomprising an amino acid sequence that is recognized by one of skill asbeing derived from the variable region of an immunoglobulin. An antibodycan consist of one or more polypeptides substantially encoded byimmunoglobulin genes, fragments of immunoglobulin genes, hybridimmunoglobulin genes (made by combining the genetic information fromdifferent animals), or synthetic immunoglobulin genes. The recognized,native, immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes and multiple D-segments andJ-segments. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. Antibodies exist as intact immunoglobulins, as a number ofwell characterized fragments produced by digestion with variouspeptidases, or as a variety of fragments made by recombinant DNAtechnology. Antibodies can derive from many different species (e.g.,rabbit, sheep, camel, human, or rodent, such as mouse or rat), or can besynthetic. Antibodies can be chimeric, humanized, or humaneered.Antibodies can be monoclonal or polyclonal, multiple or single chained,fragments or intact immunoglobulins.

As used herein, an “antibody fragment” is defined to be at least oneportion of an intact antibody, or recombinant variants thereof, andrefers to the antigen binding domain, e.g., an antigenic determiningvariable region of an intact antibody, that is sufficient to conferrecognition and specific binding of the antibody fragment to a target,such as an antigen. Examples of antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)₂, and Fv fragments, scFv antibodyfragments, linear antibodies, single domain antibodies such as sdAb(either V_(L) or V_(H)), camelid VHH domains, and multi-specificantibodies formed from antibody fragments. The term “scFv” is defined tobe a fusion protein comprising at least one antibody fragment comprisinga variable region of a light chain and at least one antibody fragmentcomprising a variable region of a heavy chain, wherein the light andheavy chain variable regions are contiguously linked via a shortflexible polypeptide linker, and capable of being expressed as a singlechain polypeptide, and wherein the scFv retains the specificity of theintact antibody from which it is derived. Unless specified, as usedherein an scFv may have the V_(L) and V_(H) variable regions in eitherorder, e.g., with respect to the N-terminal and C-terminal ends of thepolypeptide, the scFv may comprise V_(L)-linker-V_(H) or may compriseV_(H)-linker-V_(L).

As used herein, an “antigen” is defined to be a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. The skilled artisan will understand that anymacromolecule, including, but not limited to, virtually all proteins orpeptides, including glycosylated polypeptides, phosphorylatedpolypeptides, and other post-translation modified polypeptides includingpolypeptides modified with lipids, can serve as an antigen. Furthermore,antigens can be derived from recombinant or genomic DNA. A skilledartisan will understand that any DNA, which comprises a nucleotidesequences or a partial nucleotide sequence encoding a protein thatelicits an immune response therefore encodes an “antigen” as that termis used herein. Furthermore, one skilled in the art will understand thatan antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be synthesized or can be derived from a biologicalsample, or can be a macromolecule besides a polypeptide. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a fluid with other biological components.

As used herein, the terms “Chimeric Antigen Receptor” and the term “CAR”are used interchangeably. As used herein, a “CAR” is defined to be afusion protein comprising antigen recognition moieties andcell-activation elements.

As used herein, a “CAR T-cell” or “CAR T-lymphocyte” are usedinterchangeably, and are defined to be a T-cell containing thecapability of producing CAR polypeptide, regardless of actual expressionlevel. For example a cell that is capable of expressing a CAR is aT-cell containing nucleic acid sequences for the expression of the CARin the cell.

As used herein, a “costimulatory element” or “costimulatory signalingdomain” or “costimulatory polypeptide” are defined to be theintracellular portion of a costimulatory polypeptide. A costimulatorypolypeptide can be represented in the following protein families: TNFreceptor proteins, Immunoglobulin-like proteins, cytokine receptors,integrins, signaling lymphocytic activation molecules (SLAM proteins),and activating natural killer cell receptors. Examples of suchpolypeptides include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40,ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1),CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, MyD88, and thelike.

As used herein, a “Cmax” is defined to mean the maximum concentration ofa polypeptide produced by a cell after the cell is stimulated oractivated to produce the polypeptide.

As used herein, a “cytokine C_(max)” is defined to mean the maximumconcentration of cytokine produced by an immune cell after stimulationor activation to produce the cytokine.

As used herein, a “cytotoxic polypeptide C_(max)” is defined to mean themaximum concentration of cytotoxic polypeptide produced by an immunecell after stimulation or activation to produce the cytotoxicpolypeptide.

As used herein, a “destabilizing element” or a “DE” or a “Degron” areused interchangeably, and are defined to be a polypeptide sequence thatis inducibly resistant or susceptible to degradation in the cellularcontext by the addition or subtraction of a ligand, and which confersthis stability modulation to a co-translated polypeptide to which it isfused in cis.

As used herein, an “effective amount” or “therapeutically effectiveamount” are used interchangeably, and defined to be an amount of acompound, formulation, material, or composition, as described hereineffective to achieve a particular biological result.

As used herein, an “epitope” is defined to be the portion of an antigencapable of eliciting an immune response, or the portion of an antigenthat binds to an antibody. Epitopes can be a protein sequence orsubsequence that is recognized by an antibody.

As used herein, an “expression vector” and an “expression construct” areused interchangeably, and are both defined to be a plasmid, virus, orother nucleic acid designed for protein expression in a cell. The vectoror construct is used to introduce a gene into a host cell whereby thevector will interact with polymerases in the cell to express the proteinencoded in the vector/construct. The expression vector and/or expressionconstruct may exist in the cell extrachromosomally or integrated intothe chromosome. When integrated into the chromosome the nucleic acidscomprising the expression vector or expression construct will be anexpression vector or expression construct.

As used herein, an “extracellular element” is defined as the antigenbinding or recognition element of a Chimeric Antigen Receptor.

As used herein, a “hematopoietic cell” is defined to be a cell thatarises from a hematopoietic stem cell. This includes but is not limitedto myeloid progenitor cells, lymphoid progenitor cells, megakaryocytes,erythrocytes, mast cells, myeloblasts, basophils, neutrophils,eosinophils, macrophages, thrombocytes, monocytes, natural killer cells,T lymphocytes, B lymphocytes and plasma cells.

As used herein, “heterologous” is defined to mean the nucleic acidand/or polypeptide are not homologous to the host cell. For example, aconstruct is heterologous to a host cell if it contains some homologoussequences arranged in a manner not found in the host cell and/or theconstruct contains some heterologous sequences not found in the hostcell.

As used herein, an “intracellular element” is defined as the portion ofa Chimeric Antigen Receptor that resides on the cytoplasmic side of theeukaryotic cell's cytoplasmic membrane, and transmits a signal into theeukaryotic cell. The “intracellular signaling element” is that portionof the intracellular element which transduces the effector functionsignal which directs the eukaryotic cell to perform a specializedfunction.

As used herein, “RNA destabilizing element” or “RDE” are usedinterchangeably and both are defined as a nucleic acid sequence in anRNA that is bound by proteins and which protein binding changes thestability and/or translation of the RNA. Examples of RDEs include ClassI AU rich elements (ARE), Class II ARE, Class III ARE, U rich elements,GU rich elements, and stem-loop destabilizing elements (SLDE). Withoutwishing to be bound by theory, RDE's may also bind RNA stabilizingpolypeptides like HuR.

As used herein, an “RNase III substrate” is defined to be an RNAsequence motif that is recognized and cleaved by an endoribonuclease ofthe RNase III family.

As used herein, an “RNAi substrate” is defined to be an RNA sequencethat is bound and/or cleaved by a short interfering RNA (siRNA)complexed to an effector endonuclease of the Argonaute family.

As used herein, a “single chain antibody” (scFv) is defined as animmunoglobulin molecule with function in antigen-binding activities. Anantibody in scFv (single chain fragment variable) format consists ofvariable regions of heavy (V_(H)) and light (V_(L)) chains, which arejoined together by a flexible peptide linker.

As used herein, a “T-lymphocyte” or T-cell” is defined to be ahematopoietic cell that normally develops in the thymus. T-lymphocytesor T-cells include, but are not limited to, natural killer T cells,regulatory T cells, helper T cells, cytotoxic T cells, memory T cells,gamma delta T cells and mucosal invariant T cells.

As used herein, “transfected” or “transformed” or “transduced” aredefined to be a process by which exogenous nucleic acid is transferredor introduced into a host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

As used herein, a “transmembrane element” is defined as the elementbetween the extracellular element and the intracellular element. Aportion of the transmembrane element exists within the cell membrane.

Destabilizing Elements

Destabilizing elements (DE) are stability-affecting polypeptides capableof interacting with a small-molecule ligand, the presence, absence, oramount of which ligand is used to modulate the stability of theDE-polypeptide of interest. The polypeptide of interest can be animmunomodulatory polypeptide. The polypeptide of interest can also be aCAR. Binding of ligand by a DE-CAR can reduce the degradation rate ofthe DE-CAR polypeptide in the eukaryotic cell. Binding of ligand by theDE-CAR can also increase the degradation rate of the DE-CAR in theeukaryotic cell.

Exemplary destabilizing elements or DEs are described in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, both of which areincorporated by reference in their entirety for all purposes.

The ligand(s) for the DE can be selected for optimization of certainattributes for therapeutic attractiveness, for example, as described inU.S. patent application Ser. No. 15/070,352 filed on Mar. 15, 2016, andU.S. patent application Ser. No. 15/369,132 filed Dec. 5, 2016, both ofwhich are incorporated by reference in their entirety for all purposes.

RNA Control Devices

The Ribonucleic acid (RNA) control devices disclosed herein can exhibittunable regulation of gene expression, design modularity, and targetspecificity. The RNA control devices can act to rewire information flowthrough cellular networks and reprogram cellular behavior in response tochanges in the cellular environment. In regulating polypeptideexpression, the RNA control devices can serve as synthetic cellularsensors to monitor temporal and spatial fluctuations in the levels ofdiverse input molecules. RNA control devices represent powerful toolsfor constructing ligand-controlled gene regulatory systems tailored tomodulate the expression of CAR, DE-CAR, and/or Side-CAR polypeptides ofthe invention in response to specific effector molecules enabling RNAregulation of target CAR, DE-CAR, and/or Side-CAR constructs in variousliving systems.

The RNA control devices disclosed herein comprise a regulatory elementand a sensor element. The RNA control devices disclosed herein cancomprise a single element with both a regulatory and sensory function.The RNA control devices disclosed herein can comprise a regulatoryfunction and a sensory function. The RNA control devices disclosedherein can comprise a regulatory element, a sensor element, and aninformation transmission element (ITE) that functionally couples theregulatory element and the sensor element. The ITE can be based on, forexample, a strand-displacement mechanism, an electrostatic interaction,a conformation change, or a steric effect. The sensing function of theRNA control device leads to a structural change in the RNA controldevice, leading to altered activity of the acting function. Somemechanisms whereby these structural changes can occur include stericeffects, hydrophobicity driven effects (log p), electrostatically driveneffects, nucleotide modification effects (such as methylation,pseudouradination, etc.), secondary ligand interaction effects and othereffects. A strand-displacement mechanism can use competitive binding oftwo nucleic acid sequences (e.g., the competing strand and the RNAcontrol device strand) to a general transmission region of the RNAcontrol device (e.g., the base stem of the aptamer) to result indisruption or restoration of the regulatory element in response toligand binding to the sensor element.

The RNA control device can comprise a sensor element and a regulatoryelement. The sensor element can be an RNA aptamer. The RNA controldevice can have more than one sensor element. In some aspects, theregulatory element can be a ribozyme. The ribozyme can be a hammerheadribozyme. The ribozyme can also be a hairpin ribozyme, or a hepatitisdelta virus (HDV) ribozyme, or a Varkud Satellite (VS) ribozyme, a glmSribozyme, and/or other ribozymes known in the art.

The RNA control device or devices can be embedded within a DNA sequence.The RNA control device can be encoded for in messenger RNA. Multiple RNAcontrol devices can be encoded in cis with a transgene-encoding mRNA.The multiple RNA control devices can be the same and/or a mixture ofdifferent RNA control devices repeated. The nucleic acid that is used toencode the RNA control device can be repeated. By including multiple RNAcontrol devices, sensitivity and dose response may be tailored oroptimized. The multiple RNA control devices can each be specific for adifferent ligand. This can mitigate unintentional expression due toendogenously produced ligands that interact with the sensor element.

RNA Control Devices: Sensor Elements

Exemplary sensor elements are described in U.S. patent application Ser.No. 15/070,352 filed on Mar. 15, 2016, and U.S. patent application Ser.No. 15/369,132 filed Dec. 5, 2016, both of which are incorporated byreference in their entirety for all purposes. Sensor elements can bederived from aptamers. An “aptamer” is a nucleic acid molecule, such asRNA or DNA that is capable of binding to a specific molecule with highaffinity and specificity (Ellington et al., Nature 346, 818-22 (1990);and Tuerk et al., Science 249, 505-10 (1990), which are herebyincorporated by reference in their entirety for all purposes). For areview of aptamers that recognize small molecules, see Famulok, Science9:324-9 (1999), which is hereby incorporated by reference in itsentirety for all purposes.

Ligands for RNA Control Devices

RNA control devices can be controlled via the addition of exogenousligand or synthesis (or addition) of endogenous ligands with desiredbinding properties, kinetics, bioavailability, etc., for example, asdescribed in U.S. patent application Ser. No. 15/070,352 filed on Mar.15, 2016, and U.S. patent application Ser. No. 15/369,132 filed Dec. 5,2016, both of which are incorporated by reference in their entirety forall purposes.

RNA Control Devices: Regulatory Elements

The regulatory element can comprise a ribozyme, or an antisense nucleicacid, or an RNAi sequence or precursor that gives rise to a siRNA ormiRNA, or a shRNA or precursor thereof, or an RNAse III substrate, or analternative splicing element, or a transcription terminator, or aribosome binding site, or an IRES, or a polyA site. Regulatory elementsuseful in the present invention are, for example, described in U.S.patent application Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S.patent application Ser. No. 15/369,132 filed Dec. 5, 2016, both of whichare incorporated by reference in their entirety for all purposes.

RNA Destabilizing Elements

RNA destabilizing elements (RDE) are nucleic acids that affect ormaintain the stability of an RNA molecule or the translation kinetics ofan RNA molecule. Some RDEs are bound by polypeptides which destabilize(e.g., cleave) the RNA, or prevent translation, leading to loss offunction for the RNA. Some RDE binding polypeptide stabilizes the RNAincreasing the half-life of the RNA. RDEs can be used to control theexpression of a transgene, e.g., a transgene encoding a chimeric antigenreceptors. RDEs can be used with RNA control devices, DEs, and/or SideCARs to regulate the expression of a transgene. The RDEs can also beused to control expression of transgenes encoding polypeptides otherthan a CAR. Other transgenes may encode, for example, a cytokine, anantibody, a checkpoint inhibitor, a granzyme, an apoptosis inducer,complement, a cytotoxic small molecule, other cytotoxic compounds, apolypeptide for imaging, or other polypeptide that can have a desiredeffect. The RDE can control the delivery of a transgene payload.Examples of RDEs include, for example, AU rich elements, U richelements, GU rich elements, and certain stem-loop elements. ExemplaryRDEs are described in Kovarik et al., Cytokine 89:21-26 (2017); Ray etal., Nature 499:172-177 (2013); Castello et al., Cell 149:1393-1406(2012); Vlasova et al., Molc. Cell. 29:263-270 (2008); Barreau et al.,Nucl. Acids Res. vol 33, doi:10.1093/nar/gki1012 (2006); Meisner et al.,ChemBioChem 5:1432-1447 (2004); Guhaniyogi et al., Gene 265:11-23(2001), all of which are incorporated by reference in their entirety forall purposes.

The RDE can be a Class I AU rich element (dispersed AUUUA (SEQ ID NO:1)in U rich context), a Class II AU rich element (overlapping(AUUUA)_(n)), a Class III AU rich element (U-rich stretch), a stem-loopdestabilizing element (SLDE), a cytokine 3′ UTR (e.g., INF-γ, IL-2,T-cell receptor a chain, TNFα, IL-6, IL-8, GM-CSF, G-CSF etc.), and asequence of AUUUAUUUAUUUA (SEQ ID NO: 2). Khabar, WIREs RNA 2016, doi:10.1002/wrna.1368 (2016); Palanisamy et al, J. Dent. Res. 91:651-658(2012), both of which are incorporated by reference in their entiretyfor all purposes. The RDE can also be a GU rich element comprised of oneor more of, for example, UUGUU (SEQ ID NO: 3), UGGGGAU (SEQ ID NO: 4),or GUUUG (SEQ ID NO: 5). The RDE can be a U-rich element comprised ofone or more of, for example, UUUGUUU (SEQ ID NO: 6), NNU JNNUUU (SEQ IDNO: 7), UUUAUUU (SEQ ID NO: 8), UUUGUUU (SEQ ID NO: 9), UUAGA (SEQ IDNO: 10), or AGUUU (SEQ ID NO: 11). In some aspects, multiple RDEs can becombined to make a regulatory unit, for example, multiple RDEs that havethe same sequence can be arranged in a concatemer or can be arrangedwith intervening sequence in between some or all of the RDEs. The RDEsequence can be modified to increase or decrease the affinity of an RNAbinding protein(s) for the RDE. For example, an AU rich RDE can bechanged to alter the affinity of glyceraldehyde phosphate dehydrogenase(GAPDH) to the RDE. This change in affinity can alter theGAPDH-activation threshold for expression of a transgene regulated bythe RDE to which GAPDH binds.

The disclosure assigns AU # designations to some RDEs and these RDEs canbe referred to by the AU # or the gene name from which the RDE isderived. Some AU # s and the corresponding gene from which the RDE isderived include, for example, AU 1 (CD40LG), AU 2 (CSF2), AU 3 (CD247),AU 4 (CTLA4), AU 5 (EDN1), AU 6 (IL2RA), AU 7 (SLC2A1), AU 8 (TRAC), AU9 (CD274), AU 10 (Myc), AU 11 (CD19), AU 12 (IL4), AU 13 (IL5), AU 14(IL6), AU 15 (IL9), AU 16 (IL10), AU 17 (IL13), AU 18 (FOXP3), AU 19(TMEM-219), AU 20 (TMEM-219snp), AU 21 (CCR7), AU 22 (SEM-A4D), AU 23(CDC42-SE2), AU 24 (CD8), AU 27 (bGH), and AU 101 (IFNg).

The RDE can be from the 3′ UTR of a gene encoding, for example, IL-1,IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, GM-CSF, G-CSF, VEG F, PGE₂, COX-2,MMP (matrix metalloproteinases), bFGF, c-myc, c-fos, beta1-AR, PTH,interferon-gamma, MyoD, p21, Cyclin A, Cyclin B1, Cyclin D1, PAI-2, NOSHANOS, TNF-alpha, interferon-alpha, bcl-2, interferon-beta, c-jun,GLUT1, p53, Myogenin, NF-M, or GAP-43, lymphocyte antigen 96, SUPV3L1,SFtPA2, BLOC1S2, OR10A6, OR8D1, TRPT1, CIP29, EP400, PLE2, H3ST3A1,ZNF571, PPP1R14A, SPAG4L, OR10A6 and KIR3DL. Other RDEs are found in,for example, the 3′-UTRs from GLMN, AMY2B, AMY2A, AMY2A, AMY1A, TRIM33,TRIM33, TRIM33, CSRP1, PPP1R12B, KCNH1, Reticulon_4, MRPL30, Nav1.2,Tissue_factor_pathway_inhibitor, EEF1B2, CRYGB, ARMC9, RPL15, EAF2,MRPS22, MRPS22, COPB2, PDCD10, RE1-silencing_transcription_factor,Amphiregulin, AP1AR, TLR3, SKP2,Peptidylglycine_alpha-amidating_monooxygenase, TNFAIP8, Interleukin_9,PCDHA2, PCDHA12, Aldehyde_dehydrogenase_5_family,_member_A1, KCNQ5,COX7A2, Monocarboxylate_transporter_10, MLLT4, PHF10, PTPN12,MRNA_(guanine-N7-)-methyltransferase, WHSC1L1,Tricho-rhino-phalangeal_syndrome_Type_1, Interferon_alpha-1, ZCCHC6,Retinitis_pigmentosa_GTPase_regulator, MED14, CLCN5, DNA2L, OR52D1,NELL1, SLC22A25, SLC22A10, TRPC6, CACNA2D4, EPS8, CT2_(gene),Mitochondrial_ribosomal_protein_L42, TAOK3, NUPL1,Endothelin_receptor_type_B,Survival_of_motor_neuron_protein-interacting_protein_1, POLE2,Hepatic_lipase, TPSG1, TRAP1, RPS15A, HS3ST3A1, CROP_(gene),Apolipoprotein_H, GRB2, CEP76, VPS4B, Interleukin_28B, IZUMO1, FGF21,PPP1R15A, LIN7B, and CDC45-related_protein.

Still other RDEs can be found in, for example, the 3′UTRs of SCFD1,MAL2, KHSRP, IQCB1, CAMP responsive element modulator, MFAP5, SBF2,FKBP2, PDCD10, UBE2V2, NDUFAB1, Coiled-Coil_Domain_Containing_Protein,ALG13, TPTE, Enaptin, Thymopoietin, Delta-like_1, C11orf30,Actinin_alpha_4, TMEM59, SP110, Dicer, TARDBP, IFNA17, IFNA16, IFNA14,ZMYM3, Interleukin_9,_type_I, OPN1SW, THSD1, ERGIC2, CAMK2B, WDR8, FXR1,Thymine-DNA_glycosylase, Parathyroid_hormone-related_protein, OSBPL3,Ran, GYPE, AKAP4, LOC642658, L2HGDH, AKAP1, Zinc_finger_protein_334,TC2N, FKBPL, GRB14, CXorf67, CXorf66, CEP76, Gastricsin, CEP70, CYP26A1,NAA35, Aryl_hydrocarbon_receptor_nuclear_translocator, KLC4, GPR112,LARP4, NOVA1, UBE2D3, ITGA6, GPR18, MGST_type_A,RE1-silencing_transcription_factor, ASPM, ZNF452, KIR2DS4, AHSA1, TMTC4,VSX1, P16, MRPL19, CCL20, TRPT1, Hepatic_lipase, PDLIM5, CCDC53,‘CCDC55, GAPVD1, HOXB2, KCNQ5, BRCC3, GTF2IRD1, CDK5RAP3,Transcription_factor_II_B, ZEB1, IRGM, SLC39A6, RHEB, PSIP1, RPS6KA5,Urokinase receptor, GFM1, DNAJC7, Phosphoinositide-dependent_kinase-1,LMOD3, TTC35, RRP12, ATXN2, ACSM3, SOAT1, FGF8, HNRPH3, CTAGE5, POLG2,DYRK3, POLK, Cyclin-dependent_kinase_inhibitor_1C, CD137, Calmodulin_1,ZNF571, CNOT2, CRYZL1, SMC3, SMC4, SLC36A1, Decorin, HKR1, ERC1, S100A6,RIMS1, TMEM67, Mitochondrial_ribosomal_protein_L42, MECP2, RNF111,SULT1A1, MYLK3, TINAG, PRKAR1A, RGPD5, UBE2V1, SAR1B, SLC27A6, ZNF638,RAB33A, TRIOBP, MUCL1, CADPS2, MCF2L, TBCA, SLC17A3, LEO1, IFNA21,RUNX1T1, PRKD2, ATP11B, MORC2, RBM6, KLRD1, MED31, PPHLN1, HMGB2,DNA_repair_and_recombination_protein_RAD54-like, RBM9′, ARL11, HuD,SPEF2, CBLL1, SLC38A1, ‘Caspase_1’, S100G, CA1_, CELA1, PTS, ITM2B,Natriuretic_peptide_precursor_C, TRPP3, IMPDH2, DPYS, CDCA3, EFCAB6,SLIT2, SIPA1L1, FIP1L1, ATP6V1B2, HSD17B4, HSD17B7, NDUFC1, CROP, CD48,APPBP1, CD44, CD46, Histone_deacetylase_2_type_XI, Interleukin_4,Tricho-rhino-phalangeal_syndrome_Type_1, SEC61G, TRIP12, PLEKHO1,SEC61B, ST6GALNAC1, CPVL, E2F7, UTP20, E2F5, PARD3, EXOC7, HEXB,Caspase_recruitment_domain-containing_protein_8, MBD4, PPP4C, Helicase,Phosducin, SPG11, CGGBP1, PSKH1, Cathepsin_S, orexin, IMMP2L, C2orf28,Laminin, EIF3S6, LRRC41_type_XII, Cathepsin_C, HPS6, ARAF,Zinc_finger_and_BTB_domain-containing_protein_16,Sex_hormone-binding_globulin, FBLN2, Suppressor_of_cytokine_signaling_1,TMEM126A, DOM3Z, TSFM POLQ-like, DYNLT3, CDH9, EAF2, MIPEP, NDUFA12,HDAC8, MKKS, FGG, IL36G, CDCA7, CRISPLD2, Olfactomedin-like_2b, MRPL32,MRPL33, AHI1, SMARCAL1, UTP14A, SSH2, Dystonin, Contactin_6, PPFIBP1,THOC1, CNOT1, RHCE, SLC41A3, SLC2A9, SNAP23, RFX3, GNG4, MRPL40, LSR,Angiogenin, TRIP4, VRK1, COUP-TFII, FOXP2, SNX2, Nucleoporin_85, RPL37A,RPL27A, SEC62, Calcium-activated_potassium_channel_subunit_alpha-1,SMARCE1, RPL17, CEP104, CEP290, VPS29, ANXA4, Zinc_finger_protein_737,DDX59, SAP30, NEK3, Exosome_component_9,Receptor_for_activated_C_kinase_1, Peptidylprolyl_isomerase_A, TINP1,CEACAM1, DISCI, LRRTM1, POP1_Lamin_B1,SREBP_cleavage-activating_protein, COX6C, TLR_1, ARID2, LACTB, MMS22L,UBE2E3, DAP3, ZNF23, SKP2, GPR113, IRF9 Ghrelin_O-acyltransferase,NEIL3, EEF1E1, COX17, ESD_, Dentin_sialophosphoprotein, HDAC9, RFC4,CYLD, RPLP0, EIF2B3, UGT2A1, FABP7, TRIP11, PLA2G4A, AKR1C3, INTS12,MYH1, ZBTB17, MYH4, NLRP2, MECOM, MYH8, Thermogenin_receptor_2, IFI16,THYN1, RAB17, ETFA, Cystic_fibrosis_transmembrane_conductance_regulator,F13B, RAB6A, ST8SIA1, SATB2, SATB1, HMG20B, UHRF1, CNOT3,Prostaglandin_EP2_receptor, FAM65B,Peroxisome_proliferator-activated_receptor_gamma, KvLQT2, GRIK5, SHOC2,Cortactin, FANCI, KIAA1199, Kynureninase, Decoy_receptor_1, NEU3, PHF10,Methyl-CpG-binding_domain_protein_2, RABGAP1, CEP55, SF3B1, MSH5, MSH6,CREB-binding_protein, LIMS1, SLC5A4, CCNB1IP1, RNF34, SORBS2, UIMC1,SOX5, YWHAZ, ICOSLG, NOP58, Zinc_finger_protein_679, PHKB, MED13, ABCB7,COQ9, C14orf104, Zinc_finger_protein_530, KLRC2, LSM8, NBR1, PRKCD,Long-chain-aldehyde_dehydrogenase, MTSS1, Somatostatin,Ubiquitin_carboxyl-terminal_hydrolase_L5, WDR72, FERMT3,Nuclear_receptor_related-1_protein, Citrate_synthase, VPS11, KIZ,ZFYVE27, BCKDHB, Hypocretin, CACNG2, PTCH1, Carbonic_anhydrase_4,Nucleoporin_107, LDL_receptor, LEKTI, FBXO11, NDUFB3, FCHO2, CEP78,RAPGEF6, PPIL3, NIN, RAPGEF2, Growth_hormone_1, Growth_hormone_2, MNAT1,Nav1, MAP3K8, SUGT1, LAIR1, Hyaluronan-mediated_motility_receptor,MAP3K2, MPP2, TFB2M, CRB3, MPP5, CACNA1G, DLGAP2, INHBA, MAGI2, C1P29,SETDB1, Cytochrome_b5, TRPV2, Interleukin_1_receptor, HOXD8, TIMM10,ATXN2L, CLCN2, CREB1, TNIP1, CBLB, Factor_V, USP33, SON, RBBP8,SLC22A18, PTPN12, ADCY8, MYLK, KIF23, REXO2, BST1, TOP3B, COPB1, AXIN2,COPB2, TNRC6B, Guanidinoacetate_N-methyltransferase,Acyl-CoA_thioesterase_9, C4orf21, TSHB, FRS3, EPB41, Cyclin_T2, LAIR2,Nucleoporin_43, APLP2, TNFRSF19, Death-associated_protein_6,Epithelial_cell_adhesion_molecule, CLEC7A, Gephyrin, CLDND1, VPS37A,PCDHAC2, Bone_morphogenetic_protein_4, NVL, RBM33, RNF139,Sperm_associated_antigen_5, PLCB1,Glial_cell_line-derived_neurotrophic_factor, PARP4, PARP1, MAN2A1,Bone_morphogenetic_protein_1, PAX4, BCCIP, MMP7, Decoy_receptor_3,RAMP2, NCAPD3, LRRC37A, RWDD3, UBE2A, UBE2C, SLC3A1, MRPS22, CDC14A,ITSN1, POLE2, MYC-induced_nuclear_antigen, TMLHE,Glutamate_carboxypeptidase_II, GPR177, PPP2R5C, KIAA1333, RPP38, MYO1F,Farnesoid_X_receptor, Caldesmon, FBXO4, FBXO5, OPN1MW, PIGN, ARNTL2,BCAS3, C6orf58, PHTF2, SEC23A, NUFIP2, OAZ1, Osteoprotegerin, ANAPC4,ATP6V0A2, SPAM1, PSMA6, TAS2R30, RABEP1, DPM3, SLC6A15, RPS26, RPS27,RPS24, RPS20, RPS21, ARHGAP24, Catechol-O-methyl transferase, ERCC5,Transcription_initiation_protein_SPT3_homolog, OR1E1, ZNRF1, GMEB1,CCT2_GNAQ, Mucin_6, Mucin_4, LRP5, PDE9A, C2orf3, EZH2,Epidermal_growth_factor_receptor, TMTC2, PDE4A, EPH_receptor_A4, PPIB,DENND4A, ANTXR1, ANTXR2, Nucleoporin_88, SLCO1B3, COG8, RBMS1, MAP7,HIST2H2BE, AEBP2, DCLRE1A, RPL24, HNRPA2B1, RPL21, RPL23, MAPKAP1,NIPBL, ATG7, SERPINI2, GYLTL1B, ATP5G2, DIP2A, AMY2A, CEP63, TDRD7,PIEZO1, CLDN20, GRXCR1, PMEL, NIF3L1, MCC_, PCNX, TMBIM4, DUSP12,ZMYND8, GOSR1, Interferon_gamma_receptor_1, LDB3, PON3, C1D, ABCC8,COQ7, COQ6, AMELY, HAVCR1, PICALM, Sjogren_syndrome_antigen_B, PLK4,HBB, AKT1, PCDHGB7, C6orf10, UBR1, Retinoblastoma-like_protein_1, GRK6,WWC2, GRK4, INPP4B, SLC34A1, GOLGA2, MYCBP2, PTP4A2, NUCB2, MAGOH,RPP40, Alpha-2A_adrenergic_receptor, SPAG11B, Nucleoporin_205, COG1,Motile_sperm_domain_containing_3, KCNMB3,Motile_sperm_domain_containing_1, KLHL7, KCNN2, TSPAN8, GPR21,Translocator_protein, HNRNPLL, ABHD5, CAB39L, Amphiregulin, GPR1,Interleukin_18, EIF4G3, Interleukin_15, CCDC80, CD2AP, NFS1, GRB2,ULBP2, Vascular_endothelial_growth_factor_C, RPS3, TLR8,BCL2-related_protein_A1, RHOT1, Collagen, Centromere_protein_E, STMN2,HESX1, RPL7, Kalirin, PCMT1, HLA-F, SUMO2, NOX3, EP400, DNM3, EED,NGLY1, NPRL2, PLAC1, Baculoviral_IAP_repeat-containing_protein_3,C7orf31, TUBA1C, HAUS3, IFNA10, MYST4, DCHS1, SIRT4, EFEMP1, ARPC2,MED30, IFT74, PAK1IP1, DYNC1LI2, POLR2B, POLR2H, KIF3A, PRDM16, PLSCR5,PEX5, Parathyroid_hormone_1_receptor, CDC23, RBPMS, MAST1, NRD1, BAT5,BAT2, Dock11, GCSH, POF1B, USP15, POT1, MUTYH, CYP2E1, FAM122C,A1_polypeptide, Flavin_containing_monooxygenase_3, HPGD, LGALS13,MTHFD2L, Survival_motor_neuron_domain_containing_1, PSMA3, MRPS35,MHC_class_I_polypeptide-related_sequence_A, SGCE, REPS1, PPP1R12A,PPP1R12B, PABPC1, MAPK8, PDCD5, Phosphoglucomutase_3, Ubiquitin_C,GABPB2, Mitochondrial_translational_release_factor_1, PFDN4, NUB1,SLC13A3, ZFP36L1, Galectin-3, CC2D2A, GCA,Tissue_factor_pathway_inhibitor, UCKL1, ITFG3, SOS1, WWTR1, GPR84,HSPA14, GJC3, TCF7L1, Matrix_metallopeptidase_12, ISG20, LILRA3,Serum_albumin, Phosducin-like, RPS13, UTP6, HP1BP3, IL12A,HtrA_serine_peptidase_2, LATS1, BMF_, Thymosin_beta-4, B-cell_linker,BCL2L11, Coagulation_factor_XIII, BCL2L12, PRPF19, SFRS5,Interleukin_23_subunit_alpha, NRAP, 60S_ribosomal_protein_L14, C9orf64,Testin, VPS13A, DGKD, PTPRB, ATP5C1, KCNJ16, KARS, GTF2H2, AMBN, USP13,ADAMTSL1, TRO_, RTF1, ATP6V1C2, SSBP1,SNRPN_upstream_reading_frame_protein, RPS29, SNRPG, ABCC10, PTPRU,APPL1, TINF2, TMEM22, UNC45A, RPL30, PCDH7, Galactosamine-6_sulfatase,UPF3A, ACTL6A, ACTL6B, IL3RA, SDHB, Cathepsin_L2, TAS2R7, Cathepsin_L1,Pituitary_adenylate_cyclase-activating_peptide, RPN2, DYNLL1, KLK13,NDUFB3, PRPF8, SPINT2, AHSA1, Glutamate_carboxypeptidase_II, DRAP1,RNASE1, Olfactomedin-like_2b, VRK1, IKK2, ERGIC2, TAS2R16, CAMK2G,CAMK2B, Estrogen_receptor_beta, NADH_dehydrogenase, RPL19, NUCB2,KCTD13, ubiquinone, H2AFY, CEP290, PABPC1, HLA-F, DHX38, KIAA0922,MPHOSPH8, DDX59, MIB2_, ZBP1, C16orf84, UACA, C6orf142, MRPL39,Cyclin-dependent_kinase_7, Far_upstream_element-binding_protein_1,SGOL1, GTF2IRD1, ATG10, Dermcidin, EPS8L2, Decorin,Nicotinamide_phosphoribosyltransferase, CDC20, MYB, WNT5A, RBPJ,DEFB103A, RPS15A, ATP5H, RPS3, FABP1, SLC4A8, Serum_amyloid_P_component,ALAS1, MAPK1, PDCD5, SULT1A1, CHRNA3, ATXN10, MNAT1, ALG13, Ataxin_3,LRRC39, ADH7, Delta-sarcoglycan, TACC1, IFNA4,Thymic_stromal_lymphopoietin, LGTN, KIAA1333, MSH6, MYOT, RIPK5,BCL2L11, RPL27, Rnd1, Platelet_factor_4, HSD17B7, LSM8, CEP63, INTS8,CTNS, ASAHL, CELA3A, SMARCAL1, HEXB, SLC16A5, MAP3K12, FRMD6.

The RDE can be a Class I AU rich element that arises from the 3′ UTR ofa gene encoding, for example, c-myc, c-fos, beta1-AR, PTH,interferon-gamma, MyoD, p21, Cyclin A, Cyclin B1, Cyclin D1, PAI-2, orNOS HANOS. The RDE can also be a Class II AU rich element and arisesfrom the 3′ UTR of a gene encoding, for example, GM-CSF, TNF-alpha,interferon-alpha, COX-2, IL-2, IL-3, bcl-2, interferon-beta, or VEG-F.The RDE can be a Class III AU rich element that arises from the 3′ UTRof a gene encoding, for example, c-jun, GLUT1, p53, hsp 70, Myogenin,NF-M, or GAP-43. Other RDEs may be obtained from the 3′-UTRs of a T-cellreceptor subunit (α, β, γ, or δ chains), cytotoxicT-lymphocyte-associated antigen 4 (CTLA4), programmed cell death protein(PD-1), Killer-cell Immunoglobulin-like Receptors (KIR), and LymphocyteActivation Gene-3 (LAG3) and other checkpoint inhibitors. Still otherRDEs may be obtained from the 3′-UTRs of senescence-associated secretoryphenotype genes disclosed in Coppe et al., Ann. Rev. Pathol. 5:99-118(2010), which is incorporated by reference in its entirety for allpurposes (e.g., see Table 1).

The RDE can be bound by certain polypeptides including, for example, AREpoly(U) binding/degradation factor (AUF-1), tristetraprolin (TTP), humanantigen-related protein (HuR), butyrate response factor 1 (BRF-1),butyrate response factor 2 (BRF-2), T-cell restricted intracellularantigen-1 (TIA-1), TIA-1 related protein (TIAR), CUG triplet repeat, RNAbinding protein 1 (CUGBP-1), CUG triplet repeat, RNA binding protein 2(CUGBP-2), human neuron specific RNA binding protein (Hel-N1, Hel-N2),RNA binding proteins HuA, HuB and HuC, KH-type splicing regulatoryprotein (KSRP), 3-methylglutaconyl-CoA hydratase (AUH), glyceraldehyde3-phosphate dehydrogenase (GAPDH), heat shock protein 70 (Hsp70), heatshock protein 10 (Hsp10), heterogeneous nuclear ribonucleoprotein A1(hnRNP A1), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2),heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3), heterogeneousnuclear ribonucleoprotein C (hnRNP C), heterogeneous nuclearribonucleoprotein L (hnRNP L), Bcl-2 AU-rich element RNA binding protein(TINO), Poly(A) Binding Protein Interacting Protein 2 (PAIP2), IRP1,pyruvate kinase, lactate dehydrogenase, enolase, and aldolase. The RDEbinding protein also can be an enzyme involved in glycolysis orcarbohydrate metabolism, such as, for example, Glyceraldehyde PhosphateDehydrogenase (GAPDH), enolase (ENO1 or ENO3), Phosphoglycerate Kinase(PGK1), Triosephosphate Isomerase (TPI1), Aldolase A (ALDOA),Phosphoglycerate Mutase (PGAM1), Hexokinase (HK-2), or LactateDehydrogenase (LDH). The RDE binding protein can be an enzyme involvedin the Pentose Phosphate Shunt, including for example, Transketolase(TKT) or Triosephosphate Isomerase (TPI1). Additional exemplary RNAbinding proteins are those described in Castello et al., Molc. Cell63:696-710 (2016); Kovarik et al., Cytokine 89:21-26 (2017); Ray et al.,Nature 499:172-177 (2013); Castello et al., Cell 149:1393-1406 (2012);Vlasova et al., Molc. Cell. 29:263-270 (2008); Barreau et al., Nucl.Acids Res. vol 33, doi:10.1093/nar/gki1012 (2006); Meisner et al.,ChemBioChem 5:1432-1447 (2004); Guhaniyogi et al., Gene 265:11-23(2001), all of which are incorporated by reference in their entirety forall purposes.

The RDE binding protein can be TTP which can bind to RDEs including forexample, one or more of UUAUUUAUU (SEQ ID NO: 12) and AUUUA (SEQ ID NO:1), or KSRP which binds AU-rich RDEs, or Aufl which binds RDEs includingfor example, one or more of UUGA (SEQ ID NO: 13), AGUUU (SEQ ID NO: 11),or GUUUG (SEQ ID NO: 5), or CELF-1 which binds RDEs including forexample, one or more of UUGUU (SEQ ID NO: 3), or HuR which binds RDEsincluding for example, one or more of UUUAUUU (SEQ ID NO: 8), UUUAUUU(SEQ ID NO: 9), or UUUGUUU (SEQ ID NO: 6), or ESRP1 or ESRP2 which bindsRDEs including for example, one or more of UGGGGAU (SEQ ID NO: 14), orELAV which binds RDEs including for example, one or more of UUUGUUU (SEQID NO: 6). The RDE binding protein can be an enzyme involved inglycolysis, including for example, GAPDH which binds AU rich elementsincluding for example, one or more of AUUUA (SEQ ID NO: 1) elements, orENO3/ENO1 which binds RDEs including for example, one or more ofCUGCUGCUG (SEQ ID NO: 15), or ALDOA which binds RDEs including forexample, one or more of AUUGA (SEQ ID NO: 16).

In an aspect, the RDE can be combined with an RNA control device to makethe regulation by the RDE ligand inducible. For example, an RDE can beoperably linked to an RNA control device where ligand binding by the RNAcontrol device activates the regulatory element (e.g., a ribozyme orriboswitch) which inhibits the RDE (e.g., a ribozyme cleaves the RDE soRDE binding proteins no longer bind, or the riboswitch alters secondarystructure). This places transcripts with the RDE and RNA control deviceunder two types of control from the RDE, first the RDE can regulate thetranscript subject to binding of RDE binding proteins as governed byconditions in the cell, and second, the RDE control can be removed byinducing the RNA control device with ligand. When ligand is added, theRNA control device renders the RDE unavailable for binding and RDEregulation is removed. When ligand is removed, new transcripts that aretranscribed can be under the control of the RDE (as the RNA controldevice will not be activated). Alternatively, an RDE can be operablelinked to an RNA control device where ligand binding turns off theregulatory element (e.g., a ribozyme). In this example, the presence ofligand inhibits the RNA control device and transcripts can be regulatedby the RDE. When ligand is removed, the RNA control device renders theRDE unavailable for binding to RDE binding proteins and RDE regulationof the transcript is removed. The RNA control device could also cleave apolynucleotide that binds to the RDE to form a structure (e.g., a helix)that inhibits RDE proteins from binding to the RDE. In this example, theRNA control device can cleave the inhibitory polynucleotide which thendoes not bind or is inhibited for binding to the RDE. This cleavage bythe RNA control device can be stimulated by ligand binding or inhibitedby ligand binding.

Different RDEs have different kinetic parameters such as, for example,different steady expression levels, different T_(max) (time to maximalexpression level), different C_(max) (maximum expression level),different dynamic range (expression/basal expression), different AUC,different kinetics of induction (acceleration of expression rate andvelocity of expression rate), amount of expression, baseline expression,maximal dynamic range (DR_(max)), time to DR_(max), area under the curve(AUC), etc. In addition, these kinetic properties of the RDEs can bealtered by making concatemers of the same RDE, or combining differentRDEs into regulatory units. Placing RDEs under the control of anoperably linked RNA control device can also alter the kinetic propertiesof the RDE, RDE concatemer, or RDE combinations. Also, small moleculesand other molecules that affect the availability of RDE binding proteinsfor binding RDEs can be used to alter the kinetic response of an RDE,RDE concatemer, and/or RDE combinations. The kinetic response of RDEs,RDE concatemers, and/or RDE combinations can be changed using constructsthat express competitive RDEs in a transcript. Such transcripts with oneor more competing RDEs can compete for RDE binding proteins and so alterthe regulation of the desired gene by an RDE, RDE concatemer, and/or RDEcombination. These competitive RDE transcripts can bind to RDE bindingproteins reducing the amount of RDE binding protein available forbinding to the RDE, RDE concatemer, and/or RDE combination. Thus, RDEs,RDE concatemers, and/or RDE combinations can be selected and/or combinedwith other conditions (discussed above) to provide a desired kineticresponse to the expression of a transgene.

Table 2 in Example 20 shows that different RDEs (e.g., AU elements)provided different kinetics of expression. For example, different RDEs(e.g., AU elements) reached maximal induction (maximal dynamic rangealso known as fold induction) at different time points. The RDEs AU 2and AU 101 reached maximal dynamic range (DR_(max)) at day 1 and thenthe dynamic range (DR) decreased showing reduced expression compared tobasal expression. The RDEs AU 5 and AU 21 had a DR_(max) at day ¾ andthis expression was maintained out to day 8. The RDEs AU 3, AU 7, AU 10,AU 20 and AU 23 had a DR_(max) on day 6 and expression decreased on day8. The RDEs AU 19 and AU 22 had DR_(max) on day 8. The RDEs (e.g., AUelements) also had differences in the amount of expression covering arange of about 5500 fold comparing the expression of AU 7 to AU 10 (seeTable 1). Thus, RDEs (AU elements) can be selected to provide maximalrates of expression at a desired time point and to provide a desiredamount of polypeptide at that time point.

Some RNA binding proteins increase the rate of RNA degradation afterbinding to the RDE. Some RNA binding proteins decrease the rate ofdegradation of the RNA after binding to the RDE. More than one RNAbinding protein binds can bind to an RDE. In some RDE regulatory units,more than one RNA binding protein binds to more than one RDE. Binding ofone or more of the RNA binding proteins to the one or more RDEs canincrease the degradation rate of the RNA. Binding of one or more of theRNA binding proteins can decrease the degradation rate of the RNA. RNAbinding proteins that increase degradation may compete for binding to anRDE with RNA binding proteins that decrease degradation, so that thestability of the RNA is dependent of the relative binding of the two RNAbinding proteins. Other proteins can bind to the RDE binding proteinsand modulate the effect of the RNA binding protein on the RNA with theRDE. Binding of a protein to the RNA binding protein can increases RNAstability or decrease RNA stability. An RNA can have multiple RDEs thatare bound by the proteins HuR and TTP. The HuR protein can stabilize theRNA and the TTP protein can destabilize the RNA. An RNA can have atleast one RDE that interacts with the proteins KSRP, TTP and/or HuR.KSRP can destabilize the RNA and compete for binding with the HuRprotein that can stabilize the RNA. The KSRP protein can bind to the RDEand destabilizes the RNA and the TTP protein can bind to KSRP andprevent degradation of the RNA. Different proteins may be bound to thesame transcript and may have competing effects on degradation andstabilization rates. Different proteins may be bound to the sametranscript and may have cooperative effects on degradation andstabilization rates. Different proteins may be bound to the sametranscript at different times, conferring different effects ondegradation and stabilization.

The RDE can be a Class II AU rich element, and the RNA binding proteincan be GAPDH. The Class II AU rich element bound by GAPDH can beAUUUAUUUAUUUA (SEQ ID NO: 2). The Class II AU rich element and GADPH canbe used to control the expression of a transgene, a CAR, Smart CAR,DE-CAR, Smart-DE-CAR, and/or Side-CAR. The Class II AU rich element andGADPH also can be used to effect the expression of a transgene, CAR,Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side-CAR in a T-lymphocyte. TheClass II AU rich element and GADPH can be used to effect the expressionof a transgene, CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side-CAR ina CD8+ T-lymphocyte. The Class II AU rich element and GADPH can be usedto effect the expression of a transgene, CAR, Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side-CAR in a CD4+ T-lymphocyte. The Class II AUrich element and GADPH can be used to effect the expression of atransgene, CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side-CAR in anatural killer cell.

The RDE may have microRNA binding sites. The RDE can be engineered toremove one or more of these microRNA binding sites. The removal of themicroRNA binding sites can increase the on expression from a constructwith an RDE by at least 5, 10, 15, 20, 50 or 100 fold. The RDE with themicroRNA sites can be an RDE that is bound by GAPDH. The removal ofmicroRNA sites from the RDE bound by GAPDH can increase the onexpression of a construct with the GAPDH sensitive RDE by at least 5-10fold. This GAPDH control through the RDE can be used to deliver apayload at a target site. The GAPDH control can be tied to activation ofthe eukaryotic cell by a CAR that recognizes an antigen foundpreferentially at the target site.

The RDE can be the 3′-UTR of IL-2 or IFN-γ, and removal of micro-RNAsites can increase the rate of expression and/or the dynamic range ofexpression from a transgene RNA with the RDE. The RDE can be the 3′-UTRof IL-2 and the removed micro-RNA sites can be the MIR-186 sites whichdeletion increases the kinetics of expression and increases the dynamicrange of expression by about 50-fold. The RDE also can be the 3′-UTR ofIFN-γ and the micro-RNA sites removed can be the MIR-125 sites.

Chimeric Antigen Receptors

Chimeric antigen receptors (CARs) can be fused proteins comprising anextracellular antigen-binding/recognition element, a transmembraneelement that anchors the receptor to the cell membrane and at least oneintracellular element. These CAR elements are known in the art, forexample as described in patent application US20140242701, which isincorporated by reference in its entirety for all purposes herein. TheCAR can be a recombinant polypeptide expressed from a constructcomprising at least an extracellular antigen binding element, atransmembrane element and an intracellular signaling element comprisinga functional signaling element derived from a stimulatory molecule. Thestimulatory molecule can be the zeta chain associated with the T cellreceptor complex. The cytoplasmic signaling element may further compriseone or more functional signaling elements derived from at least onecostimulatory molecule. The costimulatory molecule can be chosen from4-1BB (i.e., CD137), CD27 and/or CD28. The CAR may be a chimeric fusionprotein comprising an extracellular antigen recognition element, atransmembrane element and an intracellular signaling element comprisinga functional signaling element derived from a stimulatory molecule. TheCAR may comprise a chimeric fusion protein comprising an extracellularantigen recognition element, a transmembrane element and anintracellular signaling element comprising a functional signalingelement derived from a co-stimulatory molecule and a functionalsignaling element derived from a stimulatory molecule. The CAR may be achimeric fusion protein comprising an extracellular antigen recognitionelement, a transmembrane element and an intracellular signaling elementcomprising two functional signaling elements derived from one or moreco-stimulatory molecule(s) and a functional signaling element derivedfrom a stimulatory molecule. The CAR may comprise a chimeric fusionprotein comprising an extracellular antigen recognition element, atransmembrane element and an intracellular signaling element comprisingat least two functional signaling elements derived from one or moreco-stimulatory molecule(s) and a functional signaling element derivedfrom a stimulatory molecule. The CAR may comprise an optional leadersequence at the amino-terminus (N-term) of the CAR fusion protein. TheCAR may further comprise a leader sequence at the N-terminus of theextracellular antigen recognition element, wherein the leader sequenceis optionally cleaved from the antigen recognition element (e.g., ascFv) during cellular processing and localization of the CAR to thecellular membrane.

Chimeric Antigen Receptor—Extracellular Element

Exemplary extracellular elements useful in making CARs are described,for example, in U.S. patent application Ser. No. 15/070,352 filed onMar. 15, 2016, and U.S. patent application Ser. No. 15/369,132 filedDec. 5, 2016, both of which are incorporated by reference in theirentirety for all purposes.

The extracellular element(s) can be obtained from the repertoire ofantibodies obtained from the immune cells of a subject that has becomeimmune to a disease, such as for example, as described in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, both of which areincorporated by reference in their entirety for all purposes.

The extracellular element may be obtained from any of the wide varietyof extracellular elements or secreted proteins associated with ligandbinding and/or signal transduction as described in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, U.S. Pat. Nos.5,359,046, 5,686,281 and 6,103,521, all of which are incorporated byreference in their entirety for all purposes.

Intracellular Element

The intracellular element can be a molecule that can transmit a signalinto a cell when the extracellular element of the Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,and/or Side-CAR binds to (interacts with) an antigen. The intracellularsignaling element can be generally responsible for activation of atleast one of the normal effector functions of the immune cell in whichthe Smart CAR(s), DE-CAR(s), RDE-CAR(s), Smart-RDE-CAR(s),DE-RDE-CAR(s), Smart-DE-CAR(s), Smart-DE-RDE-CAR(s) and/or Side-CAR(s)has been introduced. The term “effector function” refers to aspecialized function of a cell. Effector function of a T cell, forexample, may be cytolytic activity or helper activity including thesecretion of cytokines. Thus the term “intracellular signaling element”refers to the portion of a protein which transduces the effectorfunction signal and directs the cell to perform a specialized function.While the entire intracellular signaling domain can be employed, in manycases the intracellular element or intracellular signaling element neednot consist of the entire domain. To the extent that a truncated portionof the intracellular signaling domain is used, such truncated portionmay be used as long as it transduces the effector function signal. Theterm intracellular signaling element is thus also meant to include anytruncated portion of the intracellular signaling domain sufficient totransduce the effector function signal. Examples of intracellularsignaling elements for use in the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR of the invention include the cytoplasmic sequences of the Tcell receptor (TCR) and co-receptors that act in concert to initiatesignal transduction following antigen receptor engagement, as well asany derivative or variant of these sequences and any recombinantsequence that has the same functional capability.

Intracellular elements and combinations of polypeptides useful with oras intracellular elements are described, for example, in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S. patentapplication Ser. No. 15/369,132 filed Dec. 5, 2016, both of which areincorporated by reference in their entirety for all purposes.

Transmembrane Element and Spacer Element

The Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR may comprise a transmembrane element.The transmembrane element can be attached to the extracellular elementof the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR. The transmembrane elementcan include one or more additional amino acids adjacent to thetransmembrane region, e.g., one or more amino acid associated with theextracellular region of the protein from which the transmembrane wasderived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of theextracellular region) and/or one or more additional amino acidsassociated with the intracellular region of the protein from which thetransmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 upto 15 amino acids of the intracellular region). The transmembraneelement can be associated with one of the other elements used in theSmart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR. The transmembrane element can beselected or modified by amino acid substitution to avoid binding of suchelements to the transmembrane elements of the same or different surfacemembrane proteins, e.g., to minimize interactions with other members ofthe receptor complex. The transmembrane element can be capable ofhomodimerization with another Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR on the cellsurface. The amino acid sequence of the transmembrane element may bemodified or substituted so as to minimize interactions with the bindingelements of the native binding partner present in the same cell.

Transmembrane elements useful in the present invention are described,for example, in U.S. patent application Ser. No. 15/070,352 filed onMar. 15, 2016, and U.S. patent application Ser. No. 15/369,132 filedDec. 5, 2016, both of which are incorporated by reference in theirentirety for all purposes.

Chimeric Antigen Receptors: Side-CARs

The CARs, Smart CARs, DE-CARs, RDE-CARs, Smart-RDE-CARs, DE-RDE-CARs,Smart-DE-CARs, and/or Smart-DE-RDE-CARs can be comprised of at least twoparts which associate to form a functional CAR or DE-CAR. Theextracellular antigen binding element can be expressed as a separatepart from the transmembrane element, optional spacer, and theintracellular element of a CAR. The separate extracellular bindingelement can be associated with the host cell membrane (through a meansother than a transmembrane polypeptide). The intracellular element canbe expressed as a separate part from the extracellular element,transmembrane element, and optionally the spacer. The extracellularelement and intracellular element can be expressed separately and eachcan have a transmembrane element, and optionally a spacer. Each part ofthe CAR or DE-CAR can have an association element (“Side-CAR”) forbringing the two parts together to form a functional CAR or DE-CAR.

Side CARs, selection of Side CARs, and their use with or without atether are described, for example, in U.S. patent application Ser. No.15/070,352 filed on Mar. 15, 2016, and U.S. patent application Ser. No.15/369,132 filed Dec. 5, 2016, both of which are incorporated byreference in their entirety for all purposes.

Receptors

CARs may be used as the receptor with the cell and the RDE-transgene.CARs are described above. In addition to CARs, other receptors may beused to activate or otherwise change conditions in a cell so that atransgene under the control of an RDE is expressed. Receptors thatrecognize and respond to a chemical signal can be coupled to expressionof the transgene through the RDE. For example, ion channel-linked(ionotropic) receptors, G protein-linked (metabotropic) receptors, andenzyme-linked receptors can be coupled to the expression of thetransgene.

One class of receptor that can be coupled to transgene expression areimmune receptors such as, for example, T-cell receptors, B-cellreceptors (aka antigen receptor or immunoglobulin receptor), and innateimmunity receptors.

T-cell receptors are heterodimers of two different polypeptide chains.In humans, most T cells have a T-cell receptor made of an alpha (α)chain and a beta (β) chain have a T-cell receptor made of gamma anddelta (γ/δ) chains (encoded by TRG and TRD, respectively). Techniquesand primers for amplifying nucleic acids encoding the T-cell receptorchains from lymphocytes are well known in the art and are described in,for example, SMARTer Human TCR a/b Profiling Kits sold commercially byClontech, Boria et al., BMC Immunol. 9:50-58 (2008); Moonka et al., J.Immunol. Methods 169:41-51 (1994); Kim et al., PLoS ONE 7:e37338 (2012);Seitz et al., Proc. Natl Acad. Sci. 103:12057-62 (2006), all of whichare incorporated by reference in their entirety for all purposes. TheTCR repertoires can be used as separate chains to form an antigenbinding domain. The TCR repertoires can be converted to single chainantigen binding domains. Single chain TCRs can be made from nucleicacids encoding human alpha and beta chains using techniques well-knownin the art including, for example, those described in U.S. PatentApplication Publication No. US2012/0252742, Schodin et al., Mol.Immunol. 33:819-829 (1996); Aggen et al., “Engineering HumanSingle-Chain T Cell Receptors,” Ph.D. Thesis with the University ofIllinois at Urbana-Champaign (2010) a copy of which is found atideals.illinois.edu/bitstream/handle/2142/18585/Aggen_David.pdf?sequence=1,all of which are incorporated by reference in their entirety for allpurposes.

B-cell receptors include an immunoglobulin that is membrane bound, asignal transduction moiety, CD79, and an ITAM. Techniques and primersfor amplifying nucleic acids encoding human antibody light and heavychains are well-known in the art, and described in, for example,ProGen's Human IgG and IgM Library Primer Set, Catalog No. F2000;Andris-Widhopf et al., “Generation of Human Fab Antibody Libraries: PCRAmplification and Assembly of Light and Heavy Chain Coding Sequences,”Cold Spring Harb. Protoc. 2011; Lim et al., Nat. Biotechnol. 31:108-117(2010); Sun et al., World J. Microbiol. Biotechnol. 28:381-386 (2012);Coronella et al., Nucl. Acids. Res. 28:e85 (2000), all of which areincorporated by reference in their entirety for all purposes. Techniquesand primers for amplifying nucleic acids encoding mouse antibody lightand heavy chains are well-known in the art, and described in, forexample, U.S. Pat. No. 8,143,007; Wang et al., BMC Bioinform.7(Suppl):S9 (2006), both of which are incorporated by reference in theirentirety for all purposes. The antibody repertoires can be used asseparate chains in antigen binding domains, or converted to single chainantigen binding domains. Single chain antibodies can be made fromnucleic acids encoding human light and heavy chains using techniqueswell-known in the art including, for example, those described in Pansriet al., BMC Biotechnol. 9:6 (2009); Peraldi-Roux, Methods Molc. Biol.907:73-83 (2012), both of which are incorporated by reference in theirentirety for all purposes. Single chain antibodies can be made fromnucleic acids encoding mouse light and heavy chains using techniqueswell-known in the art including, for example, those described in Imai etal., Biol. Pharm. Bull. 29:1325-1330 (2006); Cheng et al., PLoS ONE6:e27406 (2011), both of which are incorporated by reference in theirentirety for all purposes.

Innate immunity receptors include, for example, the CD94/NKG2 receptorfamily (e.g., NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H), the 2B4receptor, the NKp30, NKp44, NKp46, and NKp80 receptors, the Toll-likereceptors (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, RP105).

G-protein linked receptors also known as seven-transmembrane domainreceptors are a large family of receptors that couple receptor bindingof ligand to cellular responses through G proteins. These G-proteins aretrimers of α, β, and γ subunits (known as Gα, Gβ, and Gγ, respectively)which are active when bound to GTP and inactive when bound to GDP. Whenthe receptor binds ligand it undergoes a conformational change andallosterically activates the G-protein to exchange GTP for bound GDP.After GTP binding the G-protein dissociates from the receptor to yield aGα-GTP monomer and a Gβγ dimer. G-protein linked receptors have beengrouped together into classes which include, for example, Rhodopsin-likereceptors, secretin receptors, metabotropic glutamate/pheromonereceptors, fungal mating pheromone receptors, cyclic AMP receptors, andfrizzled/smoothened receptors. G-protein receptors are used in a widevariety of physiological processes including detection ofelectromagnetic radiation, gustatory sense (taste), sense of smell,neurotransmission, immune system regulation, growth, cell densitysensing, etc.

Enzyme linked receptors also known as a catalytic receptor, is atransmembrane receptor, where the binding of an extracellular ligandcauses enzymatic activity on the intracellular side. Enzyme linkedreceptors have two domains joined together by a transmembrane portion(or domain) of the polypeptide. The two terminal domains are anextracellular ligand binding domain and an intracellular domain that hasa catalytic function. There are multiple families of enzyme linkedreceptors including, for example, the Erb receptor family, the glialcell-derived neurotrophic factor receptor family, the natriureticpeptide receptor family, the trk neurotrophin receptor family, and thetoll-like receptor family.

Ion channel linked receptors also known as ligand-gated ion channels arereceptors that allow ions such as, for example, Na⁺, K⁺, Ca²⁺ and Cl⁻ topass through the membrane in response to the binding of a ligand to thereceptor. There are multiple families of ligand-gated ion channelsincluding, for example, cationic cys-loop receptors, anionic cys-loopreceptors, ionotropic glutamate receptors (AMPA receptors, NMDAreceptors), GABA receptors, 5-HT receptors, ATP-gated channels, andPIP₂-gated channels.

Eukaryotic Cells

Various eukaryotic cells can be used as the eukaryotic cell of theinvention. The eukaryotic cells can be animal cells. The eukaryoticcells can be mammalian cells, such as mouse, rat, rabbit, hamster,porcine, bovine, feline, or canine. The mammalian cells can be cells ofprimates, including but not limited to, monkeys, chimpanzees, gorillas,and humans. The mammalians cells can be mouse cells, as mice routinelyfunction as a model for other mammals, most particularly for humans(see, e.g., Hanna, J. et al., Science 318:1920-23, 2007; Holtzman, D. M.et al., J Clin Invest. 103(6):R15-R21, 1999; Warren, R. S. et al., JClin Invest. 95: 1789-1797, 1995; each publication is incorporated byreference in its entirety for all purposes). Animal cells include, forexample, fibroblasts, epithelial cells (e.g., renal, mammary, prostate,lung), keratinocytes, hepatocytes, adipocytes, endothelial cells, andhematopoietic cells. The animal cells can be adult cells (e.g.,terminally differentiated, dividing or non-dividing) or embryonic cells(e.g., blastocyst cells, etc.) or stem cells. The eukaryotic cell alsocan be a cell line derived from an animal or other source.

The eukaryotic cells can be stem cells. A variety of stem cells typesare known in the art and can be used as the eukaryotic cell, includingfor example, embryonic stem cells, inducible pluripotent stem cells,hematopoietic stem cells, neural stem cells, epidermal neural crest stemcells, mammary stem cells, intestinal stem cells, mesenchymal stemcells, olfactory adult stem cells, testicular cells, and progenitorcells (e.g., neural, angioblast, osteoblast, chondroblast, pancreatic,epidermal, etc.). The stem cells can be stem cell lines derived fromcells taken from a subject.

The eukaryotic cell can be a cell found in the circulatory system of amammal, including humans. Exemplary circulatory system cells include,among others, red blood cells, platelets, plasma cells, T-cells, naturalkiller cells, B-cells, macrophages, neutrophils, or the like, andprecursor cells of the same. As a group, these cells are defined to becirculating eukaryotic cells of the invention. The eukaryotic cell canbe derived from any of these circulating eukaryotic cells. Transgenesmay be used with any of these circulating cells or eukaryotic cellsderived from the circulating cells. The eukaryotic cell can be a T-cellor T-cell precursor or progenitor cell. The eukaryotic cell can be ahelper T-cell, a cytotoxic T-cell, a memory T-cell, a regulatory T-cell,a natural killer T-cell, a mucosal associated invariant T-cell, a gammadelta T cell, or a precursor or progenitor cell to the aforementioned.The eukaryotic cell can be a natural killer cell, or a precursor orprogenitor cell to the natural killer cell. The eukaryotic cell can be aB-cell, or a B-cell precursor or progenitor cell. The eukaryotic cellcan be a neutrophil or a neutrophil precursor or progenitor cell. Theeukaryotic cell can be a megakaryocyte or a precursor or progenitor cellto the megakaryocyte. The eukaryotic cell can be a macrophage or aprecursor or progenitor cell to a macrophage.

The eukaryotic cells can be plant cells. The plant cells can be cells ofmonocotyledonous or dicotyledonous plants, including, but not limitedto, alfalfa, almonds, asparagus, avocado, banana, barley, bean,blackberry, brassicas, broccoli, cabbage, canola, carrot, cauliflower,celery, cherry, chicory, citrus, coffee, cotton, cucumber, eucalyptus,hemp, lettuce, lentil, maize, mango, melon, oat, papaya, pea, peanut,pineapple, plum, potato (including sweet potatoes), pumpkin, radish,rapeseed, raspberry, rice, rye, sorghum, soybean, spinach, strawberry,sugar beet, sugarcane, sunflower, tobacco, tomato, turnip, wheat,zucchini, and other fruiting vegetables (e.g. tomatoes, pepper, chili,eggplant, cucumber, squash etc.), other bulb vegetables (e.g., garlic,onion, leek etc.), other pome fruit (e.g. apples, pears etc.), otherstone fruit (e.g., peach, nectarine, apricot, pears, plums etc.),Arabidopsis, woody plants such as coniferous and deciduous trees, anornamental plant, a perennial grass, a forage crop, flowers, othervegetables, other fruits, other agricultural crops, herbs, grass, orperennial plant parts (e.g., bulbs; tubers; roots; crowns; stems;stolons; tillers; shoots; cuttings, including un-rooted cuttings, rootedcuttings, and callus cuttings or callus-generated plantlets; apicalmeristems etc.). The term “plants” refers to all physical parts of aplant, including seeds, seedlings, saplings, roots, tubers, stems,stalks, foliage and fruits.

The eukaryotic cells also can be algal, including but not limited toalgae of the genera Chlorella, Chlamydomonas, Scenedesmus, Isochrysis,Dunaliella, Tetraselmis, Nannochloropsis, or Prototheca. The eukaryoticcells can be fungi cells, including, but not limited to, fungi of thegenera Saccharomyces, Klyuveromyces, Candida, Pichia, Debaryomyces,Hansenula, Yarrowia, Zygosaccharomyces, or Schizosaccharomyces.

The eukaryotic cells can be obtained from a subject. The subject may beany living organisms. The cells can be derived from cells obtained froma subject. Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. T cells can be obtained from a number ofsources, including peripheral blood mononuclear cells, bone marrow,lymph node tissue, cord blood, thymus tissue, tissue from a site ofinfection, ascites, pleural effusion, spleen tissue, and tumors. Anynumber of T cell lines available in the art also may be used. T-cellscan be obtained from a unit of blood collected from a subject using anynumber of techniques known to the skilled artisan, such as Ficollseparation. Cells from the circulating blood of an individual can beobtained by apheresis. The apheresis product typically containslymphocytes, including T cells, monocytes, granulocytes, B cells, othernucleated white blood cells, red blood cells, and platelets. The cellscollected by apheresis may be washed to remove the plasma fraction andto place the cells in an appropriate buffer or media for subsequentprocessing steps. The cells can be washed with phosphate buffered saline(PBS). In an alternative aspect, the wash solution lacks calcium and maylack magnesium or may lack many if not all divalent cations. Initialactivation steps in the absence of calcium can lead to magnifiedactivation.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. Cells can be enrichedby cell sorting and/or selection via negative magnetic immunoadherenceor flow cytometry using a cocktail of monoclonal antibodies directed tocell surface markers present on the cells. For example, to enrich forCD4+ cells, a monoclonal antibody cocktail typically includes antibodiesto CD14, CD20, CD11b, CD16, HLA-DR, and CD8. It may be desirable toenrich for regulatory T cells which typically express CD4+, CD25+,CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain aspects, Tregulatory cells are depleted by anti-C25 conjugated beads or othersimilar method of selection.

T cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005, each of which is incorporated by reference in its entiretyfor all purposes.

NK cells may be expanded in the presence of a myeloid cell line that hasbeen genetically modified to express membrane bound IL-15 and 4-1BBligand (CD137L). A cell line modified in this way which does not haveMEW class I and II molecules is highly susceptible to NK cell lysis andactivates NK cells. For example, K562 myeloid cells can be transducedwith a chimeric protein construct consisting of human IL-15 maturepeptide fused to the signal peptide and transmembrane domain of humanCD8α and GFP. Transduced cells can then be single-cell cloned bylimiting dilution and a clone with the highest GFP expression andsurface IL-15 selected. This clone can then be transduced with humanCD137L, creating a K562-mb15-137L cell line. To preferentially expand NKcells, peripheral blood mononuclear cell cultures containing NK cellsare cultured with a K562-mb15-137L cell line in the presence of 10 IU/mLof IL-2 for a period of time sufficient to activate and enrich for apopulation of NK cells. This period can range from 2 to 20 days,preferably about 5 days. Expanded NK cells may then be transduced withthe anti-CD19-BB-ζ chimeric receptor.

Nucleic Acids

Also described in this disclosure are nucleic acids that encode, atleast in part, the individual peptides, polypeptides, proteins, and RNAcontrol devices described herein. The nucleic acids may be natural,synthetic or a combination thereof. The nucleic acids of the inventionmay be RNA, mRNA, DNA or cDNA.

The nucleic acids of the invention also include expression vectors, suchas plasmids, or viral vectors, or linear vectors, or vectors thatintegrate into chromosomal DNA. Expression vectors can contain a nucleicacid sequence that enables the vector to replicate in one or moreselected host cells. Such sequences are well known for a variety ofcells. The origin of replication from the plasmid pBR322 is suitable formost Gram-negative bacteria. In eukaryotic host cells, e.g., mammaliancells, the expression vector can be integrated into the host cellchromosome and then replicate with the host chromosome. Similarly,vectors can be integrated into the chromosome of prokaryotic cells.

Expression vectors also generally contain a selection gene, also termeda selectable marker. Selectable markers are well-known in the art forprokaryotic and eukaryotic cells, including host cells of the invention.Generally, the selection gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli. An exemplary selection schemecan utilize a drug to arrest growth of a host cell. Those cells that aresuccessfully transformed with a heterologous gene produce a proteinconferring drug resistance and thus survive the selection regimen. Otherselectable markers for use in bacterial or eukaryotic (includingmammalian) systems are well-known in the art.

An example of a promoter that is capable of expressing a Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR transgene in a mammalian T cell is theEF1a promoter. The native EF1a promoter drives expression of the alphasubunit of the elongation factor-1 complex, which is responsible for theenzymatic delivery of aminoacyl tRNAs to the ribosome. The EF1a promoterhas been extensively used in mammalian expression plasmids and has beenshown to be effective in driving CAR expression from transgenes clonedinto a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8):1453-1464 (2009), which is incorporated by reference in its entirety forall purposes. Another example of a promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Other constitutive promoter sequences may also be used, including, butnot limited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus promoter (MMTV), human immunodeficiency virus (HIV) longterminal repeat (LTR) promoter, MoMuLV promoter, phosphoglycerate kinase(PGK) promoter, MND promoter (a synthetic promoter that contains the U3region of a modified MoMuLV LTR with myeloproliferative sarcoma virusenhancer, see, e.g., Li et al., J. Neurosci. Methods vol. 189, pp. 56-64(2010) which is incorporated by reference in its entirety for allpurposes), an avian leukemia virus promoter, an Epstein-Barr virusimmediate early promoter, a Rous sarcoma virus promoter, as well ashuman gene promoters such as, but not limited to, the actin promoter,the myosin promoter, the elongation factor-la promoter, the hemoglobinpromoter, and the creatine kinase promoter. Further, the invention isnot limited to the use of constitutive promoters.

Inducible or repressible promoters are also contemplated for use in thisdisclosure. Examples of inducible promoters include, but are not limitedto a Nuclear Factor of Activated T-cell inducible promoter (NFAT), ametallothionein promoter, a glucocorticoid promoter, a progesteronepromoter, a tetracycline promoter, a c-fos promoter, the T-REx system ofThermoFisher which places expression from the human cytomegalovirusimmediate-early promoter under the control of tetracycline operator(s),and RheoSwitch promoters of Intrexon. Macian et al., Oncogene20:2476-2489 (2001); Karzenowski, D. et al., BioTechiques 39:191-196(2005); Dai, X. et al., Protein Expr. Purif 42:236-245 (2005); Palli, S.R. et al., Eur. J. Biochem. 270:1308-1515 (2003); Dhadialla, T_(max) S.et al., Annual Rev. Entomol. 43:545-569 (1998); Kumar, M. B, et al., J.Biol. Chem. 279:27211-27218 (2004); Verhaegent, M. et al., Annal. Chem.74:4378-4385 (2002); Katalam, A. K., et al., Molecular Therapy 13:S103(2006); and Karzenowski, D. et al., Molecular Therapy 13:S194 (2006),U.S. Pat. Nos. 8,895,306, 8,822,754, 8,748,125, 8,536,354, all of whichare incorporated by reference in their entirety for all purposes.

Expression vectors typically have promoter elements, e.g., enhancers, toregulate the frequency of transcriptional initiation. Typically, theseare located in the region 30-110 bp upstream of the start site, althougha number of promoters have been shown to contain functional elementsdownstream of the start site as well. The spacing between promoterelements frequently is flexible, so that promoter function is preservedwhen elements are inverted or moved relative to one another. In thethymidine kinase (tk) promoter, the spacing between promoter elementscan be increased to 50 bp apart before activity begins to decline.Depending on the promoter, it appears that individual elements canfunction either cooperatively or independently to activatetranscription.

The expression vector may be a bi-cistronic construct or multiplecistronic construct. The two cistrons may be oriented in oppositedirections with the control regions for the cistrons located in betweenthe two cistrons. When the construct has more than two cistrons, thecistrons may be arranged in two groups with the two groups oriented inopposite directions for transcription. Exemplary bicistronic constructsare described in Amendola et al., Nat. Biotechnol. 23:108-116 (2005),which is incorporated by reference in its entirety for all purposes. Thecontrol region for one cistron may be capable of high transcriptionactivity and the other may have low transcriptional activity underconditions of use. One or both control regions may be inducible.Examples of high transcription activity control regions include, forexample, MND, EF1-alpha, PGK1, CMV, ubiquitin C, SV40 early promoter,tetracycline-responsive element promoter, cell-specific promoters, humanbeat-actin promoter, and CBG (chicken beta-globin), optionally includingthe CMV early enhancer. Examples of low transcription activity controlregions include, for example, TRE3G (commercially sold by Clontech, atetracycline-responsive element promoter with mutations that reducebasal expression), T-REx™ (commercially sold by ThermoFisher), and aminimal TATA promoter (Kiran et al., Plant Physiol. 142:364-376 (2006),which is incorporated by reference in its entirety for all purposes),HSP68, and a minimal CMV promoter. Examples of inducible control regionsinclude, for example, NFAT control regions (Macian et al, Oncogene20:2476-2489 (2001)), and the inducible control regions described above.

The bi-cistronic construct may encode a CAR and a polypeptide that is apayload (or makes a payload) to be delivered at a target site. Exemplarypayloads are described above and below. The nucleic acid encoding theCAR can be operably linked to a strong promoter, a weak promoter, and/oran inducible promoter, and optionally, operably linked to a RNA controldevice, DE, RDE, or combination of the foregoing. The CAR can be encodedby nucleic acids in a Side-CAR format. The nucleic acid encoding thepolypeptide can be operably linked to a strong promoter, a weakpromoter, and/or an inducible promoter. The nucleic acid encoding thepolypeptide that is a payload (or makes the payload) can be under thecontrol of an RDE. The RDE may be one that responds to the activationstate of the cell through, for example, glycolytic enzymes such as, forexample, glyceraldehyde phosphate dehydrogenase (GAPDH), enolase (ENO1or ENO3), phosphoglycerate kinase (PGK1), triose phosphate isomerase(TPI1), aldolase A (ALDOA), or phosphoglycerate mutase (PGAM1). The RDEmay also be bound and regulated by other energy metabolism enzymes suchas, for example, transketolase (TKT), malate dehydrogenase (MDH2),succinyl CoA Synthetase (SUGLG1), ATP citrate lyase (ACLY), orisocitrate dehydrogenase (IDH½). The host cell can express a CAR thatbinds to its antigen at a target site in a subject. This binding ofantigen at the target site activates the cell causing the cell toincrease glycolysis which induces expression of the nucleic acidencoding the polypeptide under the control of the RDE (bound byglycolytic or other energy metabolism enzymes).

The multicistronic constructs can have three or more cistrons with eachhaving control regions (optionally inducible) and RDEs operably linkedto some or all of the transgenes. These cassettes may be organized intotwo groups that are transcribed in opposite directions on the construct.Two or more transgenes can be transcribed from the same control regionand the two or more transgenes may have IRES (internal ribosome entrysite) sequences operably linked to the downstream transgenes.Alternatively, the two or more transgenes are operably linked togetherby 2A elements as described in Plasmids 101: Multicistronic Vectorsfound at blog.addgene.org/plasmids-101-multicistrnic-vectors. Commonlyused 2A sequences include, for example, EGRGSLLTCGDVEENPGP (T2A) (SEQ IDNO: 17), ATNFSLLKQAGDVEENPGP (P2A) (SEQ ID NO: 18); QCTNYALLKLAGDVESNPGP(E2A) (SEQ ID NO: 19); and VKQTLNFDLLKLAGDVESNPGP (F2A) (SEQ ID NO: 20)all of which can optionally include the sequence GSG at the aminoterminal end. This allows multiple transgenes to be transcribed onto asingle transcript that is regulated by a 3′-UTR with an RDE (or multipleRDEs).

The bicistronic/multicistronic vector can increase the overallexpression of the two or more cistrons (versus introducing the cistronson separate constructs). The bicistronic/multicistronic construct can bederived from a lenti-virus vector. The bicistronic/multicistronicconstruct can encode a CAR and a polypeptide(s) that is encoded on atransgene(s) (e.g., a payload), and the bicistronic construct mayincrease expression of the polypeptide encoded by the transgene(s) whenthe cell is activated by the CAR.

It may be desirable to modify polypeptides described herein. One ofskill will recognize many ways of generating alterations in a givennucleic acid construct to generate variant polypeptides Such well-knownmethods include site-directed mutagenesis, PCR amplification usingdegenerate oligonucleotides, exposure of cells containing the nucleicacid to mutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques (see,e.g., Gillam and Smith, Gene 8:81-97, 1979; Roberts et al., Nature328:731-734, 1987, which is incorporated by reference in its entiretyfor all purposes). The recombinant nucleic acids encoding thepolypeptides of the invention can be modified to provide preferredcodons which enhance translation of the nucleic acid in a selectedorganism.

The polynucleotides can also include polynucleotides includingnucleotide sequences that are substantially equivalent to otherpolynucleotides described herein. Polynucleotides can have at leastabout 80%, more typically at least about 90%, and even more typically atleast about 95%, sequence identity to another polynucleotide. Thenucleic acids also provide the complement of the polynucleotidesincluding a nucleotide sequence that has at least about 80%, moretypically at least about 90%, and even more typically at least about95%, sequence identity to a polynucleotide encoding a polypeptiderecited herein. The polynucleotide can be DNA (genomic, cDNA, amplified,or synthetic) or RNA. Methods and algorithms for obtaining suchpolynucleotides are well known to those of skill in the art and caninclude, for example, methods for determining hybridization conditionswhich can routinely isolate polynucleotides of the desired sequenceidentities.

Nucleic acids which encode protein analogs or variants (i.e., whereinone or more amino acids are designed to differ from the wild typepolypeptide) may be produced using site directed mutagenesis or PCRamplification in which the primer(s) have the desired point mutations.For a detailed description of suitable mutagenesis techniques, seeSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and/or CurrentProtocols in Molecular Biology, Ausubel et al., eds, Green PublishersInc. and Wiley and Sons, N.Y (1994), each of which is incorporated byreference in its entirety for all purposes. Chemical synthesis usingmethods well known in the art, such as that described by Engels et al.,Angew Chem Intl Ed. 28:716-34, 1989 (which is incorporated by referencein its entirety for all purposes), may also be used to prepare suchnucleic acids.

Amino acid “substitutions” for creating variants are preferably theresult of replacing one amino acid with another amino acid havingsimilar structural and/or chemical properties, i.e., conservative aminoacid replacements. Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

Also disclosed herein are nucleic acids encoding Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,and/or Side-CARs. The nucleic acid encoding the Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,and/or Side-CAR can be easily prepared from an amino acid sequence ofthe specified CAR combined with the sequence of the RNA control deviceby a conventional method. A base sequence encoding an amino acidsequence can be obtained from the aforementioned NCBI RefSeq IDs oraccession numbers of GenBenk for an amino acid sequence of each element,and the nucleic acid of the present invention can be prepared using astandard molecular biological and/or chemical procedure. For example,based on the base sequence, a nucleic acid can be synthesized, and thenucleic acid of the present invention can be prepared by combining DNAfragments which are obtained from a cDNA library using a polymerasechain reaction (PCR).

The nucleic acids can be linked to another nucleic acid so as to beexpressed under control of a suitable promoter. The nucleic acid can bealso linked to, in order to attain efficient transcription of thenucleic acid, other regulatory elements that cooperate with a promoteror a transcription initiation site, for example, a nucleic acidcomprising an enhancer sequence, a polyA site, or a terminator sequence.In addition to the nucleic acid of the present invention, a gene thatcan be a marker for confirming expression of the nucleic acid (e.g. adrug resistance gene, a gene encoding a reporter enzyme, or a geneencoding a fluorescent protein) may be incorporated.

When the nucleic acid is introduced into a cell ex vivo, the nucleicacid of may be combined with a substance that promotes transference of anucleic acid into a cell, for example, a reagent for introducing anucleic acid such as a liposome or a cationic lipid, in addition to theaforementioned excipients. Alternatively, a vector carrying the nucleicacid of the present invention is also useful. Particularly, acomposition in a form suitable for administration to a living body whichcontains the nucleic acid of the present invention carried by a suitablevector is suitable for in vivo gene therapy.

Introducing Nucleic Acids into Eukaryotic Cells

A process for producing a cell expressing the Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, and/or a transgene operably linked to an RDE(s) includes astep of introducing the nucleic acid encoding a Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, and/or transgene-RDE described herein into a eukaryotic cell.This step can be carried out ex vivo. Exemplary methods for introducingnucleic acids to eukaryotic cells are described, for example, in U.S.patent application Ser. No. 15/070,352 filed on Mar. 15, 2016, and U.S.patent application Ser. No. 15/369,132 filed Dec. 5, 2016, both of whichare incorporated by reference in their entirety for all purposes.

Virus Payloads

Viruses can be used to deliver transgenes to target cells. Viruses cancarry nucleic acid constructs (e.g., transfer plasmids) as payloads andso deliver to a target cell desired nucleic acids for modification ofthe target cell genotype and/or phenotype (transiently or stably). Inmany of these transduction applications, the nucleic acid carried by thevirus does not include all of the viral genome, and often includes theviral genome signals needed for packaging the nucleic acid constructinto the virus without most or all of the rest of the viral genome. Forexample, lentiviral helper plasmid and transfer plasmids systems fortransduction of target cells are available from addgene. Other helperand transfer plasmid systems are commercially available form a number ofsources (e.g., Clontech/Takara).

When used as a payload, synthesis of viral capsids, packaging of payloadnucleic acids, and release of virus with payload nucleic acids can berestricted to the target site by timing the expression of the virusgenes for replication and coat proteins to binding of ligand by areceptor at the target site. Such control can be achieved using RDEsthat induce expression when the cell undergoes a change in metabolicstate (e.g., activation of glycolysis after receptor binding to target).This RDE control can regulate expression of master switch factors forexpression of the virus genes. For example, a transcription regulatoryfactor can be placed under the control of a suitable RDE, and the viralgenes for replication, coat proteins etc can be placed under the controlof this transcription factor. When the host cell binds to ligand at thetarget site through an appropriate receptor (e.g., a CAR) this activatesthe cell, induces expression of the transcription factor with theappropriate RDE leading to expression of the viral replication proteins,coat proteins, etc.

In helper constructs, the viral genes can be placed under the control ofa variety of transcription factors. Such transcription factors caninclude, for example, ICP4, VP16 or VP64 from Herpes Simplex, or VP30from Ebola or TFEB from Adeno associated virus. The transcription factorcan be a viral master switch such as, for example, the E1A protein ofadenovirus, or Pax5 of Epstein Barr Virus, or NS1 from influenza, NS5Afrom Hepatitis C_(max) Alternatively, the Cap and Rep functions of thevirus can be placed under the control of an appropriate RDE so that thereplication proteins and coat proteins are under the control of an RDEthat provides expression upon a change in the energy state of the cell(e.g., activation of glycolysis after receptor binding of a ligand).

Similarly, viral payload constructs can include the viral Rep, Cap andother virus genes. In these examples, an RDE can be used to regulateexpression of a master switch transcription factor for the virus(analogous to the helper constructs). This can tie synthesis of viruswith the desired nucleic acid to binding of ligand by a host cellreceptor at the target site. Examples of virus master-switchtranscription factors include E4F from adenovirus or BZLF1 from EpsteinBarr virus.

Viruses have also emerged as a highly viable treatment platform for manytypes of cancer through oncolytic virotherapy (“OVT”) (Howells et al.,Oncolytic Viruses-interaction of Virus and Tumor Cells in the Battle toEliminate Cancer, Front. Oncol., (2017);https://doi.org/10.3389/fonc.2017.00195). In these applications, virusescan effectively replicate within a host, specifically target and lysetarget cells and induce robust, long lasting target cell specificimmunity. Oncolytic virus (“OV”) can include viruses that are eithernaturally tumor-selective or can be modified to specifically target andeliminate tumor cells. Additionally, these OVs can be selectivelymodified by a variety of methods, which include, for example,insertions, which can include immune stimulators like IL-12 or a killswitch, and deletions, which can include thymidine kinase (“TK”), ofspecific genes with the aim of improving their efficacy and safetyprofiles.

OVs can be utilized that retain naturally occurring oncolytic propertiesor those that have been engineered to specifically lyse a particulartumor cell. A variety of methods can be employed to improve thespecificity of OVs, for example, taking advantage of pathways which areupregulated in tumor cells and not healthy cells and by engineering avirus that relies on such a pathway for successful infection therebyrendering the virus incapable of infecting healthy tissue. A significantadvantage of OVT lies in the fact that while viral infection candirectly lyse tumor cells, the resultant immune response will begenerated not only to viral antigens but also to tumor cell antigens. Inaddition, there are a multitude of anti-cancer genes that can beincorporated into OVs in this way in order to maximize the efficacy ofthe virus and improve the anti-tumor response generated.

There are various ways in which different viruses are able to infectcells. Some viruses, like vaccinia virus (“VV”) or Newcastle diseasevirus (“NDV”) lack specific receptors for attachment and enter cells viaendocytosis (which favors cells active metabolism, e.g., cancer cells).Other viruses have a specific receptor that they use to enter hostcells; for example, adenoviruses are able to bind CAR (a 46 kD proteinthat also mediates infection by group B coxsackieviruses), and certainRGD binding integrins, or cluster of differentiation 46 (CD46). Measlescan also use CD46 for entry, whereas herpes simplex virus (HSV) usesnectin or herpesvirus entry mediator (Dörig et al., The human CD46molecule is a receptor for measles virus (Edmonston strain), Cell,75(2):295-305 (1993); Badrinath et al., Viruses as nanomedicine forcancer, Int J Nanomedicine, 11:4835-4847 (2016)).

There are a variety of methods wherein viruses can be targeted to targetcells. These methods include, for example, exploitation of certainpathways that are aberrantly expressed in target cells to ensureengineered viruses are only capable of productive infection in cellswhich have abnormal levels of certain genes. Viral coat proteins canalso be manipulated to ensure viral infection only occurs in cells withcertain receptors, e.g., receptors found predominantly on target cells.For example, the coat/envelope proteins can be engineered to includesingle chain antibodies specific for desired target cell associatedantigens using antibody display technologies for viruses (e.g.,analogous to phage display). Alternatively, other ligands can be fusedwith coat/envelope proteins that will interact with target associatedreceptors.

Viral coat proteins, i.e., capsids, can be modified to specificallydirect viral infection to target cells. There are various ways toachieve this, for example, covering the viral surface with a polymer to“cloak” the viral receptor(s) and addition of a molecule, e.g.,epidermal growth factor (“EGF”) to the virus that binds with targetcells, e.g., some tumor cells have upregulated EGF receptor (“EGFR)expression (Morrison et al., Virotherapy of ovarian cancer withpolymer-cloaked adenovirus retargeted to the epidermal growth factorreceptor, Mol Ther., 16(2):244-251 (2008)). This particular approach notonly reduces the broad tropism conferred by the existing viral receptor,but also replaces this with a target cell specific receptor to directvirus infection to target cells, leaving healthy tissue unharmed.

An additional approach for targeting viruses uses antibodies to targetviruses to target cells. As an example of this strategy, Watkins et al.,showed that fusions between antibodies and viral coat proteins cantarget EGFR. This facilitates targeting of virus to cells expressingupregulated EGFR (Watkins et al., The ‘adenobody’ approach to viraltargeting: specific and enhanced adenoviral gene delivery, Gene Ther.,4(10):1004-1012 (1997). This antibody focused approach can also useantibody fragments such as scFv integrated into the viral capsidproteins (analogous to phage display of antibodies). For example, HSVtype-1 (“HSV-1”) relies on various glycoproteins for entry into cellsand one of these glycoproteins (“gD”) is responsible for interactionwith the viral entry receptors. An scFv targeting EGFR can be fused tothis glycoprotein, and the virus is then able to use EGFR as an entryreceptor which improves selectivity for target cell (Conner et al., Astrategy for systemic delivery of the oncolytic herpes virus HSV1716:redirected tropism by antibody-binding sites incorporated on the virionsurface as a glycoprotein D fusion protein, Gene Ther., 15(24):1579-1592(2008)). In general, the antibody fused with or otherwise associatedwith the coat proteins can be targeted to any target cell associatedantigen. For example, anti-CD19 and/or anti-CD20 antibodies can targetthe viral payload to B-cells (healthy or diseased) and anti-CD90,anti-CD117 and/or anti-CD133 antibodies can target the viral payload tocancer stem cells, including myeloma stem cells.

As recited above, differential expression of certain surface markers ontarget cells compared with healthy cells can be exploited to producetarget cell selective viruses. For example, genes can be deletedresulting in a virus that can only successfully infect certain tumortypes which over-express MEK (Veerapong et al., Systemic delivery of(gamma1)34.5-deleted herpes simplex virus-1 selectively targets andtreats distant human xenograft tumors that express high MEK activity,Cancer Res., 67(17):8301-8306 (2007)). This approach can also be usedwith HSV whereby genes can be deleted to produce a virus whichpreferentially replicates in tumor cells, which unlike healthy cells,tend to have a constitutively activated Ras pathway (Fu et al., A mutanttype 2 herpes simplex virus deleted for the protein kinase domain of theICP10 gene is a potent oncolytic virus, Mol Ther., 13(5):882-90 (2006)).As this virus initiates apoptosis in infected and bystander cells andpreferentially infects tumor cells, it can be used as oncolytic agentwith this deletion (Fu et al., An HSV-2-based oncolytic virus deleted inthe PK domain of the ICP10 gene is a potent inducer of apoptotic deathin tumor cells, Gene Ther., 14(16):1218-1225 (2007)). In addition,antibodies are other molecules can be engineered into the coat proteins(e.g., viral capsid proteins) to direct the virus to cells expressingthe surface marker (or expressing higher amounts of the surface marker).

Viruses may be modified with specific gene deletions to target the virusto target cells and inhibit infectivity in healthy cells. An example ofthis strategy is thymidine kinase (“TK”) deletion from VV. As thewild-type virus typically encodes this kinase it is able to replicate inhealthy cells, however, when the gene is deleted the virus can no longerreplicate efficiently in healthy cells. As tumor cells produce higherlevels of TK, even though the gene is deleted, the virus is still ableto replicate in these cells (McCart et al., Systemic cancer therapy witha tumor-selective vaccinia virus mutant lacking thymidine kinase andvaccinia growth factor genes, Cancer Res., 61(24):8751-8757 (2001);Hughes et al., Lister strain vaccinia virus with thymidine kinase genedeletion is a tractable platform for development of a new generation ofoncolytic virus, Gene Ther., 22(6):476-484 (2015)).

In another approach, it is possible to delete genes in the viral genometo produce a desired effect in the target cell. The deleted genes canreside in the viral construct delivered by the virus or in the targetcell. For example, the virus genome could be engineered to delete genesfor coat proteins or other factors so that the virus can lyse the targetcell (or otherwise kill the target cell) but does not create new viralparticles for infection of other cells. In target cell deletions, theconstruct delivered by the virus includes nucleic acids that cause adeletion in the genome of the target cell. Such approaches can be used,for example, to delete apoptosis-inhibiting genes.

Gene deletion strategies can also be used to improve the efficiency ofvirus delivery systems that are designed to deliver viruses to targetcells without interference from the host immune system. Viruses can bedelivered within host cells (e.g., mesenchymal stem cells), whichprovide shelter from immune attack and subvert the problem of clearanceof virus (by neutralizing antibodies) before they reach their targetcells. This method has been improved in Ad by modification of the virusto make it more infective in MSC and more efficient at killing tumorcells.

Promoters that are activated more highly in target cells versusnon-target cells can be used to control expression of a transgenecarried by the virus so that the transgene is preferentially expressedin target cells (Zhang et al., Complete eradication of hepatomas usingan oncolytic adenovirus containing AFP promoter controlling E1A and anE1B deletion to drive IL-24 expression, Cancer Gene Ther., 19(9):619-629(2012); Cheng et al., Virotherapy targeting cyclin E overexpression intumors with adenovirus-enhanced cancer-selective promoter, J Mol Med(Berl), 93(2):211-223 (2015)).

OV and other viruses can also be engineered to carry a marker (e.g., acoat protein fusion) that will enhance immune reaction and clearance ofthe virus after a subject has been exposed to virus therapy. The markercan be available on virus particles, infected cells, or both. Suchmarkers can recruit the subject's immune system to clear virus and/orinfected cells after a certain period of treatment by the virus. The OVcan also be engineered to have a kill-switch that can be activated tostop viral infection in the subject treated with the OV payload. Forexample, the OV could be engineered to place certain functions (e.g.,viral coat proteins) under the control of an inducible promoter that canbe turned off by a desired signal. Additionally, or separately, the OVcould be engineered to encode a heterologous protein under induciblecontrol that will display a protein on the surface of the infected cellthat directs the immune system to destroy the cell (e.g., a heterologousMHA that is incompatible with the host). Alternatively, the OV can beengineered to turn off coat protein expression so the infected cell stopproducing new virus.

Many different viruses or virus helper systems can be used to make theviral payload. The viral payload can be an entire virus, an engineeredvirus, or a construct with transgene(s), desired control elements, andviral packing sequences. Viruses that can be used (or viral helpersystems can be based on) AAV, VV, Adenovirus, HSV, NDV, lentivirus,retroviruses, MV, Reovirus, etc.

Multiple AAV serotypes have been identified as a promising means forgene delivery as they possess important advantages over other vectors.Specifically, they do not exhibit pathogenicity in humans and may alsoprovide significantly longer transgene expression. (Berns et al., TheCryptic Life Style of Adeno-associated Virus, Bioassays, (17):237-245(1995); Zincarelli et al., Analysis of AAV Serotypes 1-9 Mediated GeneExpression and Tropism in Mice after Systemic Injection, Mol. Thera.,16(6):1073-1080 (2008) doi: 10.1038/mt.2008.76.).

VV is a naturally oncolytic virus that has a natural tropism for tumorcells due to its sensitivity to type I interferon (Wang et al.,Disruption of Erk-dependent type I interferon induction breaks themyxoma virus species barrier, Nat Immunol., 5(12):1266-1274 (2004)). Itis a double-stranded DNA virus of the Poxviridae family. There are manydifferent strains of the virus and of these, for example, Lister, Wyethand Western Reserve strains. VV is a very promising cell killing agent(Al Yaghchi et al., Vaccinia virus, a promising new therapeutic agentfor pancreatic cancer, Immunotherapy, 7(12):1249-1258 (2015)), for manyreasons including its very short life cycle (around 8 h) and its abilityto replicate in hypoxic conditions (Hiley et al., Lister strain vacciniavirus, a potential therapeutic vector targeting hypoxic tumors, GeneTher., 17(2):281-287 (2010)). Moreover, it does not have a specificreceptor and viral fusion with the plasma membrane facilitates entry(Chung et al., A27L protein mediates vaccinia virus interaction withcell surface heparan sulfate, J Virol., 72(2):1577-1585 (1998)), whichmakes it a potential candidate for treatment of all target cell types.Furthermore, VV does not depend on the host cell for mRNA transcriptionand its entire life cycle takes place in the cytoplasm, eliminating therisk of genomic integration (Broyles S S, Vaccinia virus transcription,J Gen Virol., 84(Pt 9):2293-2303 (2003)). Modifications which canimprove the anti-tumor efficacy of VV include, for example, the additionof IL-10, which improves the oncolytic activity of VV through dampeningof anti-viral immunity (prolonging viral infection) without reducinganti-tumor immunity (Chard et al., A vaccinia virus armed withinterleukin-10 is a promising therapeutic agent for treatment of murinepancreatic cancer, Clin Cancer Res, 21(2):405-416 (2015)).

Ad received regulatory approval by the State Food and DrugAdministration in China in 2005 (Garber K., China approves world's firstoncolytic virus therapy for cancer treatment, J Natl Cancer Inst,98(5):298-300 (2006)). It is a non-enveloped, double-stranded DNA virusof the Adenoviridae family. Multiple strains of Ad exist and include,for example, Ad5. Ad5 is has been widely studied and there exist amultitude of various modifications in the art, which have been shown toimprove efficacy. For example, the combination of p53 addition tosuppress tumor growth with GM-CSF addition to induce the apoptoticpathway elicits a synergistic effect which is effective in combatinghepatocellular cancer stem cells (Lv et al., 11R-P53 and GM-CSFexpressing oncolytic adenovirus target cancer stem cells with enhancedsynergistic activity, J Cancer, 8(2):199-206 (2017)).

Ad can be improved by the incorporation of a short-hairpin RNA, whichfunctions to downregulate Dicer (an endoribonuclease which has a role inprocessing virus-associated RNA). Downregulation of this proteininhibits the destruction of viral RNA and allows Ad to replicateefficiently and therefore improves the efficacy of this OV (Machitani etal., Enhanced oncolytic activities of the telomerase-specificreplication-competent adenovirus expressing short-hairpin RNA againstDicer, Mol Cancer Ther., 16(1):251-9 (2017)).

Gene silencing techniques can also be used to downregulate geneexpression in target cells. For example, EphA3 expression can bedownregulated by siRNA targeting this gene expressed from a virus. Thisapproach can be used to downregulate the expression of other desiredgenes in a target cell.

A T-cell immune response to oncolytic virus infection can be efficaciousin killing target cells as cytotoxic T-cells kill the virus infectedtarget cells (e.g., Li et al., The efficacy of oncolytic adenovirus ismediated by T-cell responses against virus and tumor in Syrian hamstermodel, Clin Cancer Res, 23(1):239-249 (2017)).

In addition, the virus payloads described herein can include any of theother payloads encoded by a transgene that are described herein.Including for example, checkpoint inhibitors, granzymes, apoptosisinducing polypeptides, etc.

The present invention further provides a therapeutic approach whereintwo or more antigenically distinct viruses are employed. One method, forexample, is achieved by employing VV after first administeringadenoviral therapy. In this approach, it has been observed that theincreased efficacy was dependent on T-cell activity (Tysome et al., Anovel therapeutic regimen to eradicate established solid tumors with aneffective induction of tumor-specific immunity, Clin Cancer Res.,18(24):6679-6689 (2012)). Another example includes, sequential deliveryof oncolytic Ad and Newcastle Disease Virus (“NDV”), which are bothengineered to express an immuno-stimulatory cytokine which leads tosignificant anti-tumor responses even though when administered alone,each virus demonstrates limited efficacy against tumors (Nistal-Villanet al., Enhanced therapeutic effect using sequential administration ofantigenically distinct oncolytic viruses expressing oncostatin M in aSyrian hamster orthotopic pancreatic cancer model, Mol Cancer, 14:210(2015)).

The oncolytic potential of viruses can be increased by combiningoncolytic therapy with induction of the autophagy pathway. The autophagypathway is involved in viral antigen presentation and therefore itsupregulation can increase presentation of virally deliveredtumor-associated antigens (“TAAs”) at the cell surface to induce a morepotent anti-tumor immune response than with antigen delivery alone(Klein et al., Critical role of autophagy in the processing ofadenovirus capsid-incorporated cancer-specific antigens, PLoS One,11(4):e0153814 (2016)).

A potential barrier to viral spread in tumor masses is the interstitialmatrix (including extracellular DNA). Viruses can be modified to encodeproteins which will degrade the interstitial matrix along withexpression of DNase I to degrade extracellular DNA, therefore allowingmore efficient spread of viruses throughout target cell masses(Tedcastle et al., Actin-resistant DNAse I expression from oncolyticadenovirus enadenotucirev enhances its intratumoral spread and reducestumor growth, Mol Ther., 24(4):796-804 (2016)).

Viruses can also be exploited as carriers for drugs using more than onemethod. For example, it has recently been reported that electrostaticattraction between viral capsid and the drug molecules themselves was anefficient way to deliver anti-cancer drugs which would then actsynergistically with the oncolytic adenoviral therapy (Garofalo et al.,Oncolytic adenovirus loaded with L-carnosine as novel strategy toenhance the antitumor activity, Mol Cancer Ther., 15(4):651-60 (2016)).

Herpes simplex virus type-1 a double-stranded DNA virus belonging to theHerpesviridae family can also be used. HSV-1 was the first virus whereinthe TK gene mutation was engineered. In 1991, Martuza et al.demonstrated that human glioblastoma cells can be destroyed by HSV-1carrying a mutation in the TK region and this was observed in cellculture as well as in nude mice (Martuza et al., Experimental therapy ofhuman glioma by means of a genetically engineered virus mutant, Science,252(5007):854-856 (1991)). Much effort has since been put into makingHSV more active against tumor cells and safer for normal cellsculminating in the approval of Talimogene laherparepvec (“T-Vec”), anengineered HSV-1 for the treatment of melanoma in 2015 (Coffin R.,Interview with Robert Coffin, inventor of T-Vec: The first oncolyticimmunotherapy approved for the treatment of cancer, Immunotherapy,8(2):103-106 (2016)). T-Vec has two viral gene deletions (one in theγ34.5 gene and one in the α47 gene) and it has the human GM-CSF geneinserted in place of the deleted γ34.5 gene. The function of γ34.5 is toprevent infected cells from switching off protein synthesis upon viralinfection.

Another HSV that can be used as a payload is G47Δ, a third generationHSV-1 with three different mutations. NV1020 virus is an HSV that can beused as a payload. NV1020 virus based on the R7020 construct developedby Meignier et al. (Meignier et al., In vivo behavior of geneticallyengineered herpes simplex viruses R7017 and R7020: construction andevaluation in rodents, J Infect Dis, 158(3):602-14 (1988)). NV1020 virushas deletions in the ICP0 and ICP4 gene regions and has only one copy ofthe γ34.5 gene. Moreover, the α4 promoter, which controls TK expression,has been inserted, making the virus sensitive to common drugs, forexample, acyclovir.

The Newcastle Disease Virus (NDV) can also be used a viral payload. Ithas been found that NDV can effectively kill a variety of tumor celltypes and that this activity occurs by induction of immunogenic celldeath which in turn leads to adaptive anti-tumor immunity (Koks et al.,Newcastle disease virotherapy induces long-term survival andtumor-specific immune memory in orthotopic glioma through the inductionof immunogenic cell death, Int J Cancer, 136(5):E313-25 (2015)). Certainstrains of NDV can induce apoptosis in target cells (Lazar et al., Theoncolytic activity of Newcastle disease virus NDV-HUJ on chemoresistantprimary melanoma cells is dependent on the proapoptotic activity of theinhibitor of apoptosis protein livin, J Virol, 84(1):639-46 (2010)), andthat the apoptosis pathway stimulated in infected tumor cells isp53-independent and perhaps triggered by endoplasmic stress (Fabian etal., p53-independent endoplasmic reticulum stress-mediated cytotoxicityof a Newcastle disease virus strain in tumor cell lines, J Virol,81(6):2817-30 (2007)). It has been discovered that apoptosis of infectedcells occurs predominantly via the intrinsic mitochondrial pathway andis caspase dependent (Elankumaran et al., Newcastle disease virus exertsoncolysis by both intrinsic and extrinsic caspase-dependent pathways ofcell death, J Virol, 80(15):7522-34 (2006)). This induction of apoptosisresults in tumor cell death.

As well as arming viruses with immune stimulators, other therapeuticgenes can increase anti-target cell effects of viral therapies. Forexample, NDV engineered to encode TNF receptor Fas shows greateroncolytic effect as Fas is responsible for increased apoptosis ofinfected cells via both the intrinsic and extrinsic apoptosis pathways,thereby increasing cell death and in turn anti-tumor efficacy(Cuadrado-Castano et al., Enhancement of the proapoptotic properties ofNewcastle disease virus promotes tumor remission in syngeneic murinecancer models, Mol Cancer Ther, 14(5):1247-58 (2015)). NDV's naturallyoncolytic properties can also be augmented by arming the virus withGM-CSF (Janke et al., Recombinant Newcastle disease virus (NDV) withinserted gene coding for GM-CSF as a new vector for cancer immunogenetherapy, Gene Ther, 14(23):1639-49 (2007)).

Retroviruses are yet another virus useful as a payload. A method hasbeen developed whereby retroviral particles, which retain theirreplicative ability, can be delivered and selectively replicate only incells which are undergoing proliferation (e.g., tumor cells) and arecompromised in their ability to trigger innate immune responses. Theability of these particles to integrate into the host genome andreplicate without causing lysis of the cell makes them efficient andlong-lasting producers of the therapeutic proteins they are deliveringwithout the consequences of productive viral infection (Logg et al.,Retroviral replicating vectors in cancer, Methods Enzymol, 507:199-228(2012)).

As is the case for traditional oncolytic viral therapy, this method canbe designed using a variety of retroviruses and with the addition ofvarious therapeutic genes. For example, suicide genes which trigger celldeath can be delivered to tumor cells via particles from variousleukemia viruses (Lu et al., Replicating retroviral vectors foroncolytic virotherapy of experimental hepatocellular carcinoma, OncolRep, 28(1):21-26 (2012)).

As well as delivering therapeutic genes, replicating retroviral vectorscan also be used to enhance the response to anti-target cell drugtherapy. For example, delivery of an activator of a therapeutic drug byreplication competent retroviral vector resulted in significantanti-tumor effect and prolonged survival time in a murine model ofmalignant mesothelioma (Kawasaki et al., Replication-competentretrovirus vector-mediated prodrug activator gene therapy inexperimental models of human malignant mesothelioma, Cancer Gene Ther,18(8):571-8 (2011)). These vectors can also be delivered within a“gutted” Ad genome and the outcome of this combination is improvedtransfer efficiency of the retroviral genome into the tumor tissue andtherefore increased production of the therapeutic gene (Kubo et al.,Adenovirus-retrovirus hybrid vectors achieve highly enhanced tumortransduction and antitumor efficacy in vivo, Mol Ther., 19(1):76-82(2011)).

MV is another useful virus payload. MV is a single-stranded, negativesense enveloped RNA virus of the Paramyxoviridae family. There are anumber of receptors that can be utilized by MV to successfully infectcells including CD150, CD46, and nectin-4. Of these, CD46 has beenattributed to increased specificity of MV to certain target cells thatexpress increased levels of this receptor compared with healthy cells.This increased expression leads to increased levels of cell lysis uponinfection of target cells compared with healthy tissue (Anderson et al.,High CD46 receptor density determines preferential killing of tumorcells by oncolytic measles virus, Cancer Res, 64(14):4919-26 (2004)).Selectivity can also be increased by engineering a MV which is blindedto its usual receptors and redirected to recognize specific target cellmarkers as target antigens (Nakamura et al., Rescue and propagation offully retargeted oncolytic measles viruses, Nat Biotechnol, 23(2):209-14(2005)). Another method of increasing MV selectivity is to engineermiRNA sensitive viruses which can only successfully infect cells inwhich certain miRNAs are downregulated (e.g., cancer cells). Forexample, a virus has been developed which shows sensitivity to threehost miRNAs through insertion of specific miRNA target sites into theviral genome, rendering the virus incapable of infecting healthy cellswhich express one or more of these miRNAs but still able to infectspecific cancer cells which have downregulated levels of these miRNAs(Baertsch et al., MicroRNA-mediated multi-tissue de-targeting ofoncolytic measles virus, Cancer Gene Ther., 21(9):373-80 (2014)).

Reovirus is a double-stranded, non-enveloped RNA virus of the Reoviridaefamily and is considered a naturally occurring OV. Reoviruses arethought to selectively infect tumor cells because their oncolyticfunctions depend on the activation of the Ras pathway (Strong et al.,The molecular basis of viral oncolysis: usurpation of the Ras signalingpathway by reovirus, EMBO J, 17(12):3351-62 (1998)), which tends to beupregulated in transformed cells. Two additional viruses that can bepayloads include, for example, coxsackievirus and echovirus.

Manipulating Target Cell Genes to Increase Virus Targeting

Target cell genes can play a role in the targeting of virus to targetcells. For example, it was discovered by Cuddington et al., that acertain virus (Bovine herpesvirus-1) are better able to infect cellswhich have increased levels of certain proteins (e.g., KRAS tumor cells)(Cuddington et al., Permissiveness of human cancer cells to oncolyticbovine herpesvirus 1 is mediated in part by KRAS activity, J Virol,88(12):6885-95 (2014)).

The aberrant expression of components of the Raf/MEK/ERK pathway intarget cells can also have an effect on the regulation of Ad receptorand therefore levels of viral infectivity. As this pathway tends to beupregulated in some potential target cells (e.g., tumor cells) comparedwith healthy cells, it can have a significant effect on viral therapy.

As well as mutations, regulation of certain genes using microRNA canalso be used to enhance viral specificity for target cells. For example,using an miRNA which is downregulated in target cells (such as let-7a intumor cells) to control expression of an essential viral gene in VV(such as B5R which increases both pathogenicity and oncolytic activity)results in a virus that can only express sufficient amounts of B5R incells which have low levels of let-7a expression, i.e., tumor cells(Hikichi et al., MicroRNA regulation of glycoprotein B5R in oncolyticvaccinia virus reduces viral pathogenicity without impairing itsantitumor efficacy, Mol Ther., 19(6):1107-15 (2011)).

Another gene found in tumor cells that can influence virus therapy isVEGF. It has been demonstrated that VEGF-A increases VV internalizationand in turn replication levels (Hiley et al., Vascular endothelialgrowth factor A promotes vaccinia virus entry into host cells viaactivation of the Akt pathway, J Virol., 87(5):2781-90 (2013)).Therefore, VV can take advantage of the increased expression of VEGF bytarget cells to increase delivery of therapeutic genes which in turnincreases the efficacy and potency of the treatment.

In addition to this, it has been discovered that the increase in VEGFexpression upon infection with VV leads to upregulation of PRD1-BF1 (atranscription repressor), which increases sensitivity of tumor vascularendothelial cells to infection with vaccinia via repression of type-1interferon anti-viral signaling. This increase in viral tropism allowsthe OV to spread through the tumor more efficiently and thereforeincreases the efficacy of this oncolytic therapy (Arulanandam et al.,VEGF-mediated induction of PRD1-BF1/Blimp1 expression sensitizes tumorvasculature to oncolytic virus infection, Cancer Cell, 28(2):210-24(2015)). This natural repression of interferon signaling highlights thepotential of using interferon inhibitors to increase the efficacy ofoncolytic viral therapy (Stewart et al., Inhibitors of the interferonresponse enhance virus replication in vitro, PLoS One, 9(11):e112014(2014)).

It has also been demonstrated that a properly functioning hostinterferon response pathway is a critical factor in measles infection ofmalignant pleural mesothelioma. It was seen that in cell lines, there isa correlation between sensitivity of cells to measles infection and aninability of the cell to elicit a full interferon response in thepresence of MV (Achard et al., Sensitivity of human pleural mesotheliomato oncolytic measles virus depends on defects of the type I interferonresponse, Oncotarget, 6(42):44892-904 (2015)). It has also beendemonstrated that VV infection is greatly increased throughdownregulation of c-Jun NH2-terminal kinase (JNK). Inhibition of the JNKsignaling cascade leads to lower levels of double-stranded RNA dependentprotein kinase, which in turn permits increased replication of VVgenomes (Hu et al., JNK-deficiency enhanced oncolytic vaccinia virusreplication and blocked activation of double-stranded RNA-dependentprotein kinase, Cancer Gene Ther., 15(9):616-24 (2008)).

Another gene found in tumor cells (CEACAM6), also has an effect ononcolytic viral therapy. This tumor-associated gene has variousfunctions, which include, for example, a role in promotion of tumoradhesion and invasion among other factors (Duxbury et al.,Overexpression of CEACAM6 promotes insulin-like growth factor I-inducedpancreatic adenocarcinoma cellular invasiveness, Oncogene,23(34):5834-42 (2004)).

Eukaryotic Cells Expressing the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR

The cell expressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE can be cells in which a nucleic acid encoding a Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE is introduced andexpressed.

Eukaryotic cells can bind to a specific antigen via the CAR, DE-CAR,and/or Side-CAR polypeptide causing the CAR, DE-CAR, and/or Side-CARpolypeptide to transmit a signal into the eukaryotic cell, and as aresult, the eukaryotic cell can be activated. The activation of theeukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR can bevaried depending on the kind of a eukaryotic cell and the intracellularelement of the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR, and can be confirmedbased on, for example, release of a cytokine, improvement of a cellproliferation rate, change in a cell surface molecule, or the like as anindex. For example, release of a cytotoxic cytokine (a tumor necrosisfactor, lymphotoxin, etc.) from the activated cell causes destruction ofa target cell expressing an antigen. In addition, release of a cytokineor change in a cell surface molecule stimulates other immune cells, forexample, a B cell, a dendritic cell, a NK cell, and/or a macrophage.

A eukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE can be used as a therapeutic agent to treat adisease. This therapeutic agent can comprise the eukaryotic cellexpressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE as anactive ingredient, and may further comprise a suitable excipient.Examples of the excipient include pharmaceutically acceptable excipientsfor the composition. The disease against which the eukaryotic cellexpressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE isadministered is not particularly limited as long as the disease showssensitivity to the eukaryotic cell. Examples of diseases of theinvention include a cancer (blood cancer (leukemia), solid tumor(ovarian cancer) etc.), an inflammatory disease/autoimmune disease(asthma, eczema), hepatitis, and an infectious disease, the cause ofwhich is a virus such as influenza and HIV, a bacterium, or a fungus,for example, tuberculosis, MRSA, VRE, and deep mycosis. An autoimmunedisease (e.g., pemphigus vulgaris, lupus erythematosus, rheumatoidarthritis) can be treated with a eukaryotic cell expressing a Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE that binds to theimmune proteins that cause the autoimmune disease. For example, theeukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE can target cells that make an antibody which causes theautoimmune disease. The eukaryotic cell expressing the Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE could targetT-lymphocytes which cause the autoimmune disease.

Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE can be used as atherapeutic agent to treat an allergy. Such therapeutic agents cancomprise the eukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE as an active ingredient, and may further comprise asuitable excipient. Examples of the excipient include pharmaceuticallyacceptable excipients for the composition. Examples of allergies thatcan be treated with the eukaryotic cell expressing the Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE include, for example,allergies to pollen, animal dander, peanuts, other nuts, milk products,gluten, eggs, seafood, shellfish, and soy. The eukaryotic cellexpressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE can targetcells that make an antibody which causes the allergic reaction against,for example, pollen, animal dander, peanuts, other nuts, milk products,gluten, eggs, seafood, shellfish, and soy. The targeted cells can be oneor more of B-cells, memory B-cells, plasma cells, pre-B-cells, andprogenitor B-cells. The eukaryotic cell expressing the Smart CAR,DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE can targetT-lymphocytes which cause the allergic reaction against, for example,pollen, animal dander, peanuts, other nuts, milk products, gluten, eggs,seafood, shellfish, and soy. The Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE can bind to the idiotypic determinant of theantibody or T-cell receptor.

The eukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,and/or transgene-RDE can be administered for treatment of a disease orcondition. These eukaryotic cells can be utilized for prevention of aninfectious disease after bone marrow transplantation or exposure toradiation, donor lymphocyte transfusion for the purpose of remission ofrecurrent leukemia, and the like. The therapeutic agent comprising theeukaryotic cell expressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/ortransgene-RDE can be an active ingredient and can be administeredintradermally, intramuscularly, subcutaneously, intraperitoneally,intranasally, intraarterially, intravenously, intratumorally, or into anafferent lymph vessel, by parenteral administration, for example, byinjection or infusion, although the administration route is not limited.

The Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE can be used with aT-lymphocyte that has aggressive anti-tumor properties, such as thosedescribed in Pegram et al, CD28z CARs and armored CARs, 2014, Cancer J.20(2):127-133, which is incorporated by reference in its entirety forall purposes. The RNA control device can be used with an armored CAR,DE-CAR, and/or Side-CAR polypeptide in a T-lymphocyte.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention may comprise aSmart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE expressing cell, e.g.,a plurality of Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, and/or transgene-RDE expressingcells, as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions of the present invention arein one aspect formulated for intravenous administration.

Pharmaceutical compositions may be administered in a manner appropriateto the disease to be treated (or prevented). The quantity and frequencyof administration will be determined by such factors as the condition ofthe patient, and the type and severity of the patient's disease,although appropriate dosages may be determined by clinical trials.

Suitable pharmaceutically acceptable excipients are well known to aperson skilled in the art. Examples of the pharmaceutically acceptableexcipients include phosphate buffered saline (e.g. 0.01 M phosphate,0.138 M NaCl, 0.0027 M KCl, pH 7.4), an aqueous solution containing amineral acid salt such as a hydrochloride, a hydrobromide, a phosphate,or a sulfate, saline, a solution of glycol or ethanol, and a salt of anorganic acid such as an acetate, a propionate, a malonate or a benzoate.An adjuvant such as a wetting agent or an emulsifier, and a pH bufferingagent can also be used. The pharmaceutically acceptable excipientsdescribed in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J.1991) (which is incorporated herein by reference in its entirety for allpurposes) can be appropriately used. The composition can be formulatedinto a known form suitable for parenteral administration, for example,injection or infusion. The composition may comprise formulationadditives such as a suspending agent, a preservative, a stabilizerand/or a dispersant, and a preservation agent for extending a validityterm during storage.

A composition comprising the eukaryotic cells described herein as anactive ingredient can be administered for treatment of, for example, acancer (blood cancer (leukemia), solid tumor (ovarian cancer) etc.), aninflammatory disease/autoimmune disease (pemphigus vulgaris, lupuserythematosus, rheumatoid arthritis, asthma, eczema), hepatitis, and aninfectious disease the cause of which is a virus such as influenza andHIV, a bacterium, or a fungus, for example, a disease such astuberculosis, MRSA, VRE, or deep mycosis, depending on an antigen towhich a CAR, DE-CAR, and/or Side-CAR polypeptide binds.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, intranasally, intraarterially,intratumorally, into an afferent lymph vessel, by intravenous (i.v.)injection, or intraperitoneally. In one aspect, the T cell compositionsof the present invention are administered to a patient by intradermal orsubcutaneous injection. In one aspect, the T-cell compositions of thepresent invention are administered by i.v. injection. The compositionsof T-cells may be injected directly into a tumor, lymph node, or site ofinfection. The administration can be done by adoptive transfer.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). A pharmaceutical composition comprising the eukaryotic cellsdescribed herein may be administered at a dosage of 10⁴ to 10⁹ cells/kgbody weight, in some instances 10⁵ to 10⁶ cells/kg body weight,including all integer values within those ranges. A eukaryotic cellcomposition may also be administered multiple times at these dosages.Eukaryotic cells can also be administered by using infusion techniquesthat are commonly known in immunotherapy (see, e.g., Rosenberg et al.,New Eng. J. of Med. 319:1676, 1988, which is incorporated by referencein its entirety for all purposes).

Uses of Eukaryotic Cells

Nucleic acids encoding Smart CAR(s), DE-CAR(s), RDE-CAR(s),Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s), Smart-DE-RDE-CAR(s),Side-CAR(s), and/or transgene-RDE(s) can be used to express CAR, DE-CAR,Side-CAR, and/or transgene polypeptides in eukaryotic cells. Theeukaryotic cell can be a mammalian cell, including for example humancells or murine cells. The eukaryotic cells may also be, for example,hematopoietic cells including, e.g., T-cells, natural killer cells,B-cells, or macrophages.

T-cells (e.g., CD4+ or CD8+) or natural killer cells can be engineeredwith a polynucleotide encoding a Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR. Ligand for the RNA control device, DE, or Side CAR is added tothe T-cells (e.g., CD4+ or CD8+) or natural killer cells can be added inincreasing amounts to obtain a desired amount of effector function. Thedesired amount of effector function can be an optimized amount ofeffector function with a known amount (and/or density) of target antigenon target cells. Effector function can be target cell killing,activation of host immune cells, cytokine secretion, production ofgranzymes, production of apoptosis inducing ligands, production of otherligands that modulate the immune system, etc. The effector function canbe secretion of cytokines such as, for example, IL-2, IFN-γ, TNF-α,TGF-β, and/or IL-10. Effector function can be the killing of targetcells. Target cells can be killed with granzymes. Target cells can beinduced to undergo apoptosis. Eukaryotic cells with CARs can kill targetcells through apoptosis and granzymes.

The RDE, DE, RNA control device, or Side CAR regulatory element can beused to control expression of a transgene. This transgene expression candeliver a payload at a target site. These transgenes can also be carriedby viral constructs, or viruses when the payload is a virus. Expressionof the transgene can cause a desired change in the eukaryotic cell. AnRDE regulated by GAPDH can be used for payload delivery, and theeukaryotic cell (e.g., T-cell, natural killer cell, B-cell, macrophage,dendritic cell, or other antigen presenting cell) can be activated(e.g., by a CAR) when it reaches the target site. Upon activation of theeukaryotic cell at the target site through the CAR, the cell inducesglycolysis and the GAPDH releases from the RDE allowed payloadexpression and delivery. The target site can be a tumor or infection andthe transgene could encode a cytokine, a chemokine, an antibody, acheckpoint inhibitor, a granzyme, an apoptosis inducer, complement, anenzyme for making a cytotoxic small molecule, an enzyme that cleavespeptides or saccharides (e.g., for digesting a biofilm), other cytotoxiccompounds, or other polypeptides that can have a desired effect at thetarget site. Checkpoint inhibitors include agents that act at immunecheckpoints including, for example, cytotoxic T-lymphocyte-associatedantigen 4 (CTLA4), programmed cell death protein (PD-1), Killer-cellImmunoglobulin-like Receptors (KIR), and Lymphocyte Activation Gene-3(LAG3). Examples of checkpoint inhibitors that may be used as payloadsinclude, for example, Nivolumab (Opdivo), Pembrolizumab (Keytruda),Atezolizumab (Tecentriq), Ipilimumab (Yervoy), Lirilumab, andBMS-986016. Nivolumab, Atezolizumab and Pembrolizumab act at thecheckpoint protein PD-1 and inhibit apoptosis of anti-tumor immunecells. Some checkpoint inhibitors prevent the interaction between PD-1and its ligand PD-L1. Ipilimumab acts at CTLA4 and prevents CTLA4 fromdownregulating activated T-cells in the tumor. Lirilumab acts at KIR andfacilitates activation of Natural Killer cells. BMS-986016 acts at LAG3and activates antigen-specific T-lymphocytes and enhances cytotoxic Tcell-mediated lysis of tumor cells. Cytokines can include, for example,IL-2, IL-12, IL-15, IL-18, IFN-γ, TNF-α, TGF-β, and/or IL-10. Cytotoxicagents can include, for example, granzymes, apoptosis inducers,complement, or a cytotoxic small molecule. The payload delivered at atarget site (e.g., non-tumor target site) can be a factor that protectsthe target site such as, for example, an anti-inflammatory, a factorthat attracts T-regulatory cells to the site, or cytokines or otherfactors that cause suppression and reduction in immune activity. Thepayload can be an enzyme that cleaves peptides or saccharides, forexample hyaluronidase, heparanase, metalloproteinases and otherproteinases which can be used, for example, to digest an undesiredbiofilm. The payload can be an imaging agent that allows the target siteto be imaged. The payload may be a polypeptide that can be imageddirectly, or it can be a polypeptide that interacts with a substrate tomake a product that can be imaged, imaging polypeptides include, forexample, thymidine kinase (PET), dopamine D2 (D2R) receptor, sodiumiodide transporter (NIS), dexoycytidine kinase, somatostatin receptorsubtype 2, norepinephrine transporter (NET), cannabinoid receptor,glucose transporter (Glut1), tyrosinase, sodium iodide transporter,dopamine D2 (D2R) receptor, modified haloalkane dehalogenase,tyrosinase, β-galactosidase, and somatostatin receptor 2. These reporterpayloads can be imaged using, for example, optical imaging, ultrasoundimaging, computed tomography imaging, optical coherence tomographyimaging, radiography imaging, nuclear medical imaging, positron emissiontomography imaging, tomography imaging, photo acoustic tomographyimaging, x-ray imaging, thermal imaging, fluoroscopy imaging,bioluminescent imaging, and fluorescent imaging. These imaging methodsinclude Positron Emission Tomography (PET) or Single Photon EmissionComputed Tomography (SPECT).

Multiple systems are envisioned for use that can kill target cellsdirectly. These include, for example, the introduction of a viral or abacterial gene into target cells. This approach turns a non-toxicpro-drug to a toxic one. There are systems that have been extensivelyinvestigated: the cytosine deaminase gene (“CD”) of Escherichia coli,which converts the pro-drug 5-Fluorocytosine (“5-FC”) to 5-Fluorouracil(“5-FU”); and the herpes simplex virus thymidine kinase gene (“HSV-tk”),which converts ganciclovir (“GCV”) to ganciclovir monophosphate,converted by the cancer cells' enzymes to ganciclovir triphosphate. TheHSV-tk/GCV system useful in killing tumor cells directly, involvesadenoviral transfer of HSV-tk to tumor cells, with the subsequentadministration of ganciclovir. Specifically, recombinantreplication-defective adenovirus is employed to transfer the thymidine,HSV-tk, into hepatocellular carcinoma (“HCC”) cells to confersensitivity to ganciclovir. Three useful HCC cell lines include, forexample, Hep3B, PLC/PRF/5 and HepG2, which can efficiently infect, invitro, by a recombinant adenovirus carrying lacZ reporter gene(“Ad-CMVlacZ”). Expression of HSV-tk in HCC cells infected with arecombinant adenovirus carrying HSV-tk gene (“AdCMVtk”) inducessensitivity to ganciclovir in a dose-dependent manner (Qian et al.,Induction of sensitivity to ganciclovir in human hepatocellularcarcinoma cells by adenovirus-mediated gene transfer of herpes simplexvirus thymidine kinase, Hepatology, 22:118-123 (1995))https://doi.org/10.1002/hep.1840220119.

Thymidine kinase can be used with PET reporter probes such as, forexample, [¹⁸F]9-(4-[¹⁸F]-fluoro-3-hydroxymethylbutyl)-guanine, afluorine-18-labelled penciclovir analogue, which when phosphorylated bythymidine kinase (TK) becomes retained intracellularly, or is 5-(76)Br-bromo-2′-fluoro-2′-deoxyuridine. The relevant reporter probes foreach of the PET reporters are well known to the skilled artisan. Anexemplary reporter probe for dopamine D2 (D2R) receptor is3-(2′-[¹⁸F]fluoroethyl)spiperone (FESP) (MacLaren et al., Gene Ther.6(5):785-91 (1999)). An exemplary reporter probe for the sodium iodidetransporter is ¹²⁴I, which is retained in cells following transport bythe transporter. An exemplary reporter probe for deoxycytidine kinase is2′-deoxy-2′-¹⁸F-5-ethyl-1-β-d-arabinofuranosyluracil (¹⁸F-FEAU). Anexemplary reporter probe for somatostatin receptor subtype 2 is ¹¹¹In-,^(99m/94m)Tc-, ⁹⁰Y-, or ¹⁷⁷Lu-labeled octreotide analogues, for example⁹⁰Y-, or ¹⁷⁷Lu-labeled DOTATOC (Zhang et al., J Nucl Med. 50(suppl2):323 (2009)); ⁶⁸Ga-DOTATATE; and ¹¹¹In-DOTABASS (see. e.g., Brader etal., J Nucl Med. 54(2):167-172 (2013), incorporated herein byreference). An exemplary reporter probe for norepinephrine transporteris ¹¹C-m-hydroxyephedrine (Buursma et al., J Nucl Med. 46:2068-2075(2005)). An exemplary reporter probe for the cannabinoid receptor is¹¹C-labeled CB2 ligand, ¹¹C-GW405833 (Vandeputte et al., J Nucl Med.52(7):1102-1109 (2011)). An exemplary reporter probe for the glucosetransporter is [¹⁸F]fluoro-2-deoxy-d-glucose (Herschman, H. R., Crit RevOncology/Hematology 51:191-204 (2004)). An exemplary reporter probe fortyrosinase is N-(2-(diethylamino)ethyl)-¹⁸F-5-fluoropicolinamide (Qin etal., Sci Rep. 3:1490 (2013)). Other reporter probes are described in theart, for example, in Yaghoubi et al., Theranostics 2(4):374-391 (2012),incorporated herein by reference.

An exemplary photoacoustic reporter probe for β-galactosidase is5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) (Li et al., J BiomedOpt. 12(2):020504 (2007)). Exemplary X-ray reporter includes, amongothers, somatostatin receptor 2, or other types of receptor basedbinding agents. The reporter probe can have a radiopaque label moietythat is bound to the reporter probe and imaged, for example, by X-ray orcomputer tomography. Exemplary radiopaque label is iodine, particularlya polyiodinated chemical group (see, e.g., U.S. Pat. No. 5,141,739), andparamagnetic labels (e.g., gadolinium), which can be attached to thereporter probe by conventional means. Optical imaging agents include,for example, a fluorescent polypeptide. Fluorescent polypeptidesinclude, for example, green fluorescent protein from Aequorea victoriaor Renilla reniformis, and active variants thereof (e.g., bluefluorescent protein, yellow fluorescent protein, cyan fluorescentprotein, etc.); fluorescent proteins from Hydroid jellyfishes, Copepod,Ctenophora, Anthrozoas, and Entacmaea quadricolor, and active variantsthereof; and phycobiliproteins and active variants thereof. The opticalimaging agent can also be a bioluminescent polypeptide. These include,for example, aequorin (and other Ca⁺² regulated photoproteins),luciferase based on luciferin substrate, luciferase based onCoelenterazine substrate (e.g., Renilla, Gaussia, and Metridina), andluciferase from Cypridina, and active variants thereof.

Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, Side-CAR and/or universal-CARs can be designed toinclude receptors against antigens that are of bacterial, fungal orviral origin. Because Smart CAR(s), DE-CAR(s), RDE-CAR(s),Smart-RDE-CAR(s), DE-RDE-CAR(s), Smart-DE-CAR(s), Smart-DE-RDE-CAR(s)and/or Side-CAR(s) can be utilized to fight infections, which are asource of toxicity in immunocompromised patients, such anti-pathogenSmart CAR(s), DE-CAR(s), RDE-CAR(s), Smart-RDE-CAR(s), DE-RDE-CAR(s),Smart-DE-CAR(s), Smart-DE-RDE-CAR(s) and/or Side-CAR(s) can be used inconjunction Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR T-cell therapy specificfor a TAA.

A eukaryotic cell can bind to a specific antigen via the CAR, DE-CAR,and/or Side-CAR polypeptide causing the CAR, DE-CAR, and/or Side-CARpolypeptide to transmit a signal into the eukaryotic cell, and as aresult, the eukaryotic cell can be activated and so express anappropriate RDE-transgene. The activation of the eukaryotic cellexpressing the Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, and/or Side-CAR is varied depending on thekind of a eukaryotic cell and the intracellular element of the SmartCAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR,Smart-DE-RDE-CAR, and/or Side-CAR. The eukaryotic cell can express a RDEtranscript that poises the cell for effector function upon stimulationof the eukaryotic cell through a Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, and/orSide-CAR.

A eukaryotic cell expressing the RDE-transgene or RDE transcript, andoptionally, a Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, T-cell receptor, B-cellreceptor, innate immunity receptor and/or other receptor or targetingpolypeptide can be used as a therapeutic agent to treat a disease. Thetherapeutic agent can comprise the eukaryotic cell expressing theRDE-transgene or RDE transcript, and optionally, a Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, T-cell receptor, B-cell receptor, innate immunity receptorand/or other receptor or targeting polypeptide as an active ingredient,and may further comprise a suitable excipient. Examples of the excipientinclude pharmaceutically acceptable excipients for the composition. Thedisease against which the eukaryotic cell expressing the RDE-transgeneor RDE transcript, and optionally, a Smart CAR, DE-CAR, RDE-CAR,Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR,T-cell receptor, B-cell receptor, innate immunity receptor and/or otherreceptor or targeting polypeptide is administered is not particularlylimited as long as the disease shows sensitivity to the eukaryotic celland/or the product of the RDE-transgene.

Examples of diseases that can be treated include a cancer (blood cancer(leukemia), solid tumor (ovarian cancer) etc.), an inflammatorydisease/autoimmune disease (asthma, eczema), hepatitis, and aninfectious disease, the cause of which is a virus such as influenza andHIV, a bacterium, or a fungus, for example, tuberculosis, MRSA, VRE, anddeep mycosis, other immune mediated diseases such as neurodegenerativediseases like Alzheimer's or Parkinson's, and metabolic diseases likediabetes. A receptor (e.g., a CAR) can target the eukaryotic cell to thediseased cell(s) and when the receptor binds to its target at thediseased cell(s) the receptor can send a signal into the eukaryotic cellleading to expression of the RDE-transgene. The RDE-transgene encodes apolypeptide that is useful in treating or killing the diseased cell(s).A cancer and/or solid tumor can be treated with a eukaryotic cellexpressing receptor that binds to a tumor associated (or cancerassociated) antigen, such as those described above. When the receptorbinds to the tumor associated antigen the receptor sends a signal intothe cell that causes the RDE-transgene to be expressed (e.g., the signaleffects an RDE binding protein leading to expression of theRDE-transcript). The RDE-transcript can encode a polypeptide thatactivates the eukaryotic cell so that the eukaryotic cell treats thecancer and/or the RDE-transcript encodes a polypeptide that itselftreats the cancer (e.g., a cytotoxic polypeptide).

An autoimmune disease (e.g., pemphigus vulgaris, lupus erythematosus,rheumatoid arthritis, multiple sclerosis, Crohn's disease) can betreated with a eukaryotic cell expressing a RDE-transgene or RDEtranscript, and optionally, a Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR,Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, T-cell receptor,B-cell receptor, innate immunity receptor and/or other receptor ortargeting polypeptide that binds to the immune proteins associated withthe autoimmune disease. The receptor or targeting polypeptide cantrigger expression of the RDE-transgene that encodes a polypeptideuseful in treating the autoimmune disease (e.g., the polypeptide canregulate the cells causing the autoimmune disease or kill these cells).The eukaryotic cell expressing the RDE-transgene or RDE transcript, andreceptor or targeting polypeptide can target cells that make an antibodyinvolved with the autoimmune disease (e.g., the RDE-transgene can encodea polypeptide that kills the antibody producing cells or that inhibitsthe production of antibody by these cells). The eukaryotic cellexpressing the RDE-transgene or RDE transcript, and receptor ortargeting polypeptide can target T-lymphocytes involved with theautoimmune disease (e.g., the RDE-transgene can encode a polypeptidethat kills the target T-lymphocytes or that regulates the activity ofthe T-lymphocytes).

Eukaryotic cells expressing the RDE-transgene or RDE transcript, andoptionally, a Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, T-cell receptor, B-cellreceptor, innate immunity receptor and/or other receptor or targetingpolypeptide can be used as a therapeutic agent to treat an allergy.Examples of allergies that can be treated include, for example,allergies to pollen, animal dander, peanuts, other nuts, milk products,gluten, eggs, seafood, shellfish, and soy. The eukaryotic cellexpressing the RDE-transgene or RDE transcript, and receptor ortargeting polypeptide can target cells that make an antibody whichcauses the allergic reaction against, for example, pollen, animaldander, peanuts, other nuts, milk products, gluten, eggs, seafood,shellfish, and soy. The targeted cells can be one or more of B-cells,memory B-cells, plasma cells, pre-B-cells, and progenitor B-cells.Targeted cells can also include T-lymphocytes which cause the allergicreaction against, for example, pollen, animal dander, peanuts, othernuts, milk products, gluten, eggs, seafood, shellfish, and soy.Eukaryotic cells expressing the RDE-transgene or RDE transcript, andreceptor or targeting polypeptide can bind to the idiotypic determinantof the antibody or T-cell receptor.

The eukaryotic cell expressing the RDE-transgene or RDE transcript, andoptionally, a Smart CAR, DE-CAR, RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR,DE-RDE-CAR, Smart-DE-RDE-CAR, Side-CAR, T-cell receptor, B-cellreceptor, innate immunity receptor and/or other receptor or targetingpolypeptide can be administered for treatment of a disease or condition.For example, the eukaryotic cell can be utilized to treat an infectiousdisease. The eukaryotic cell can express a receptor or targetingpolypeptide that binds to an antigen found on the infectious diseasecausing agent or a cell infected with such an agent. The receptor ortargeting polypeptide binds the antigen associated with the infectiousdisease and sends a signal into the eukaryotic cell that leads toexpression of the RDE-transgene. The RDE-transgene encodes a productthat can activate the eukaryotic cell for treating the infectiousdisease (e.g., the eukaryotic cell can produce a cytotoxic polypeptideor a cytokine that activates immune cells). The RDE-transgene can alsoencode a polypeptide that itself is a cytotoxic polypeptide or acytokine. The eukaryotic cell can also be utilized for prevention of aninfectious disease (used prophylactically), for example, after bonemarrow transplantation or exposure to radiation, donor lymphocytetransfusion for the purpose of remission of recurrent leukemia, and thelike.

The therapeutic agent comprising the eukaryotic cell expressing theRDE-transgene or RDE transcript, and optionally, a Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, T-cell receptor, B-cell receptor, innate immunity receptorand/or other receptor or targeting polypeptide as an active ingredientcan be administered intradermally, intramuscularly, subcutaneously,intraperitoneally, intranasally, intraarterially, intravenously,intratumorally, or into an afferent lymph vessel, by parenteraladministration, for example, by injection or infusion, although theadministration route is not limited.

The RDE-transgene or RDE transcript, and optionally, Smart CAR, DE-CAR,RDE-CAR, Smart-DE-CAR, Smart-RDE-CAR, DE-RDE-CAR, Smart-DE-RDE-CAR,Side-CAR, T-cell receptor, B-cell receptor, innate immunity receptorand/or other receptor or targeting polypeptide can be used with aT-lymphocyte that has aggressive anti-tumor properties, such as thosedescribed in Pegram et al, CD28z CARs and armored CARs, 2014, Cancer J.20(2):127-133, which is incorporated by reference in its entirety forall purposes. The RDE transcript can encode a polypeptide that causesaggressive anti-tumor properties in the T-lymphocyte.

A transgene, a CAR, DE-CAR, and/or Side CAR polypeptides can becontrolled by an RDE from the 3′-UTR of the gene encoding IL-2 or the3′-UTR of IFN-γ. These RDEs can be modified to inactivate microRNA sitesfound in the RDE. Using these control elements makes expression of theCAR, DE-CAR, Side-CAR, and/or transgene sensitive to changes in theglycolytic state of the host cell through the interaction of the RDEwith glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When the hostcell is in a quiescent state a large proportion of the GAPDH is notinvolved in glycolysis and is able to bind to the RDE resulting inreduced translation of the transcript encoding the CAR, DE-CAR,Side-CAR, and/or transgene polypeptides. When the host cell is inducedto increase glycolysis, e.g., by providing the host cells with glucose,or other small molecules that will increase glycolytic activity, GAPDHbecomes enzymatically active and is not able to bind to the RDE. Thereduction in GAPDH binding to the RDE increases translation of thetranscripts (e.g., by increasing half-life of the transcript and/or byincreasing the translation rate) encoding the CAR, DE-CAR, Side-CAR, orother transgene. The glycolytic activity of GAPDH can be increased byincreasing the amount and/or activity of triose isomerase. The host cellcan be induced to over-express a recombinant triose isomerase, and thisover-expression increases the glycolytic activity of GAPDH. A glycolysisinhibitor can be added to decrease expression of the transcript with theRDE. Such glycolysis inhibitors include for example, dimethylfumarate(DMF), rapamycin, 2-deoxyglucose, 3-bromophyruvic acid, iodoacetate,fluoride, oxamate, ploglitazone, dichloroacetic acid, quinones, or othermetabolism inhibitors such as, for example, dehydroepiandrosterone.Expression from the RDE controlled transcript can be increased by theaddition of GAPDH (or other RDE binding protein) inhibitor that inhibitsbinding of the RDE by GAPDH (or other RDE binding protein). Such GAPDHinhibitors include, for example, CGP 3466B maleate or Heptelidic acid(both sold by Santa Cruz Biotechnology, Inc.), pentalenolactone, or3-bromopyruvic acid.

Constructs encoding transcripts with RDEs can be expressed in eukaryoticcells to bind to RDE binding proteins and so reduce the ability of thoseRDE binding proteins to interact with native transcripts in the cell.The recombinant transcripts can compete for binding of RDE bindingproteins and this can reduce the inhibition and/or activation of nativetranscripts within the eukaryotic cell by the RDE binding proteins. Theconstructs encoding transcripts with the RDEs can be used in this way tochange when and how native transcripts are expressed in the eukaryoticcell. The eukaryotic cell can be a T-cell, natural killer cell, orB-cell and the recombinant transcript has RDEs that are shared withcytokine or cytotoxic transcripts (e.g., in their 3′ untranslatedregions). The recombinant transcript can compete for binding with theRDE binding proteins (e.g., GAPDH and/or other glycolytic enzymesdescribed above) that regulate expression of the cytokine or cytotoxicpolypeptide and change the threshold (e.g., glycolysis activity forGAPDH) needed to express the cytokine or cytotoxic polypeptide. This canbe used to create super T-cell (aka Angry T-cells or Hornet T-cells)that will secrete higher amounts of cytokines and/or cytotoxic proteins(greater C_(max)) in response to stimulation of the immune cell (e.g.,through a CAR or T-cell receptor). T-cells can be reprogrammed with arecombinant transcript encoding an RDE from an IL-2 transcript so thatwhen the T-cell is stimulated by its T-cell receptor it produces moreIL-2 and other effector polypeptides with faster kinetics. Thesereprogrammed T-cells can also produce other inflammatory cytokines andcytotoxic polypeptides (e.g., granzymes and/or perforins) in largeramounts and with faster kinetics. Reprogramming T-cells and naturalkiller cells into such Angry/Hornet states can be useful for treatingdisease and disorders, including, for example, tumors, other cancers,and infectious diseases.

RDEs can be used to reduce CAR expression in immune cells until thoseimmune cells are activated by target or at a desired time. This canresult in expression of the CAR at desired times for therapeutic effectwhile reducing the systemic exposure of a subject to the CAR. Thereduced systemic exposure can reduce and/or inhibit the development ofan immune response against the CAR as the subject's immune system willsee less CAR over time.

Some neural degenerative diseases and syndromes are associated withinflammation, as are a number of other non-neural diseases andsyndromes. Such inflammation associated diseases can be treated, atleast in part, by providing a subject with small molecules (or othermolecules) that increase the availability of inhibitory RDE bindingproteins within immune cells. Such small molecules include, for example,glycolysis inhibitors (e.g., dimethylfumarate (DMF), rapamycin,2-deoxyglucose, 3-bromophyruvic acid, iodoacetate, fluoride, oxamate,ploglitazone, dichloroacetic acid), other metabolic inhibitors (e.g.,dehydroepiandrosterone), etc. For example, glycolytic inhibitors reduceglycolysis in the cell and can increase the amount of free GAPDH (notinvolved in glycolysis) for binding to RDEs reducing the expression ofthese transcripts. A number of inflammatory gene products in immunecells (e.g., gene products that activate the immune system) areregulated by RDEs that can bind GAPDH. Decreasing glycolysis increasesthe amount of free GAPDH for RDE binding, increases the amount of GAPDHbound to the RDEs of these inflammatory genes and reduces the expressionof these inflammatory genes. Inflammatory genes include proinflammatorycytokines such as, for example, IL-1, TNF-α, INF-g, and GM-CSF. Thesecytokines have 3′-UTRs with RDEs that can bind RDE binding proteins,including GAPDH, to regulate their expression. The increased GAPDH canbind to these RDEs and decrease the expression of these proinflammatorycytokines. Reduced expression of proinflammatory cytokines could reduceactivity of the immune system in these subjects reducing inflammation.The reduction in inflammation can have positive therapeutic effectsalleviating symptoms and/or treating the underlying disease state inthese inflammation related neural diseases, as well as in otherinflammation associated diseases and syndromes.

RDEs (e.g., AU elements) can be selected to provide maximal expressionat a desired time point and to provide a desired amount of polypeptideat that time point. RDEs can also be selected to provide a desired areaunder the curve for a polypeptide. As shown in Table 2 of Example 20,different RDEs (e.g., AU elements) reached maximal rates of expressionat different times. Also as shown in Table 1, different RDEs provideddifferent amounts of expression with different profiles over timeproviding different AUC. Using these RDEs in combination with differenttransgenes allows temporal programming of when the different transgenesreach maximal rates of expression in relation to one another followingactivation of a cell. In addition, using different RDEs one can programthe transgenes to express a desired amount of transgene encodedpolypeptide and/or a desired amount of AUC or exposure to thepolypeptide encoded by the transgene. Thus, RDEs can be used to providecontrol that produces desired amounts of different transgenepolypeptides at a different (or the same) desired times.

This temporal control can be used to provide desired timing for theproduction of different transgene polypeptides within a cell. Using thistemporal control, a cell can be programmed to express a first transgenethat alters the state of the cell so that is prepared to be affected bythe polypeptide of a second transgene that is expressed at a later time.For example, the first expressed polypeptide could induce the cell tomake and store cytotoxic polypeptides (e.g., granzymes and/or perforins)and the second expressed polypeptide could be involved in the release ofthe cytotoxic polypeptides. Another example of temporal expressioninvolves it use to program a cell to undergo changes (e.g.,differentiation or changing a state of the cell) that requires temporalexpression of two or more gene products. RDEs can be used to mimic thistemporal expression allowing one to control when the cell changes itsstate or differentiates (e.g., programmed differentiation of stemcells). In a stem cell example, the temporal and induction control canbe used to program a stem cell to differentiate when (and where) it isdesired to have the stem cell differentiate into a desired cell type.

The temporal control can also be used to provide desired timing of theproduction of different transgene polypeptides outside of the cell.Using this temporal control, a cell can be activated and secrete a firsttransgene polypeptide that conditions and/or alters a target cell sothat the target cell is prepared to be acted upon by a polypeptidesexpressed at later time from a second transgene. For example, the firstpolypeptide could induce a target cell to express a receptor on thetarget cell surface (e.g., FasR, Her2, CD20, CTLA-4, PD-L1, etc.) or apolypeptide in the cell. The first transgene could also induce the cellto secrete a factor that induces the target cell to change its state(e.g., the first transgene could induce the cell to secrete CpG whichcauses the target cell to express OX40 on the target cell surface). Thesecond transgene that reaches maximal rate of expression at a later timecan encode a polypeptide that acts on the induced surface receptor(e.g., FasL, Herceptin, Rituximab, Ipilimumab, Nivolumab, anti-OX40antibody, etc.). The temporal and induction control can also be used tochange the state or differentiation of a target cell by providing to thetarget cell polypeptides in a timed manner where the first polypeptideinduces the target cell to alter its state (e.g., differentiation) sothat it can be acted upon by the second polypeptide (etc. for additionaltransgene polypeptides which reach maximal rate of expression at latertimes).

Some examples of diseases and payloads that can be treated using RDEs(Gold elements) with different kinetic parameters (e.g., an RDE thatgives rapid expression early after activation of the cell followed by arapid decline in expression or an RDE that delays expression after cellactivation for 2-3 days) include the following: DLL3 positive cancers(such as IDH1mut gliomas, melanoma, and SCLC) using an anti-DLL3 CAR anda payload of one or more of anti-4-1BB antibody, anti-CD11b antibody,anti-CTLA4 antibody, anti-IL1b antibody, a BiTE, CCL2, anti-CXCR4antibody, anti-CXCL12 antibody, HAC, heparinase, hyaluronidase, Hsp60,Hsp70, IL-2, IL-12, IL-15, IL-18, INFγ, miRNA (e.g., mir155), and/orCD40 ligand. CD19 positive lymphomas (e.g., NHL) using an anti-CD19 CARand a payload of IL-12, or one or more of anti-4-1BB antibody,anti-CD11b antibody, anti-CTLA4 antibody, anti-IL1b antibody, a BiTE,CCL2, anti-CXCR4 antibody, anti-CXCL12 antibody, HAC, heparinase,hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA (e.g.,mir155), and/or CD40 ligand. AML with onco-CD43 (sialylation mutant)using an anti-onco-CD43 CAR that recognizes the mutated sialylation anda payload of one or more of anti-CXCL12 antibody, anti-anti-CXCR4antibody, or IL-12, or one or more of anti-4-1BB antibody, anti-CD11bantibody, anti-CTLA4 antibody, anti-IL1b antibody, a BiTE, CCL2, HAC,heparinase, hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA(e.g., mir155), and/or CD40 ligand. PSCA positive prostate cancer,bladder cancer or pancreatic cancer using an anti-PSCA CAR and a payloadof heparinase or IL-12, or one or more of anti-4-1BB antibody,anti-CD11b antibody, anti-CTLA4 antibody, anti-IL1b antibody, a BiTE,CCL2, anti-CXCR4 antibody, anti-CXCL12 antibody, HAC, hyaluronidase,Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA (e.g., mir155), and/orCD40 ligand. Triple negative breast cancer with a CAR that recognizescancer testis antigen, misfolded or mutant EGFR (associated with triplenegative breast cancer), and/or folate receptor alpha peptide and apayload of IL-12 or one or more of anti-4-1BB antibody, anti-CD11bantibody, anti-CTLA4 antibody, anti-IL1b antibody, a BiTE, CCL2,anti-CXCR4 antibody, anti-CXCL12 antibody, HAC, heparinase,hyaluronidase, Hsp60, Hsp70, IL-2, IL-15, IL-18, INFγ, miRNA (e.g.,mir155), and/or CD40 ligand.

The inventions disclosed herein will be better understood from theexperimental details which follow. However, one skilled in the art willreadily appreciate that the specific methods and results discussed aremerely illustrative of the inventions as described more fully in theclaims which follow thereafter. Unless otherwise indicated, thedisclosure is not limited to specific procedures, materials, or thelike, as such may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

EXAMPLES Example 1. Control of T-Cell Effector Activity with an RDE-CAR

A RDE Car is made using the third generation anti-CD19 CAR cassettedescribed in WO 2012/079000, which is hereby incorporated-by-referencein its entirety for all purposes), and the 3′-UTR of the gene encodingIL-2 (NCBI Reference Sequence Number: NM_000586.3), which is herebyincorporated by reference in its entirety for all purposes). A nucleicacid encoding the IL-2 3′-UTR is engineered into the anti-CD19 CARcassette in an appropriate expression vector. The IL-2, 3′-UTR sequenceused was:

(SEQ ID NO: 21) taattaagtgatcccacttaaaacatatcaggccttctATTTATTTAaatATTTAaattttatATTTAttgttgaatgtatggtttgctacctattgtaactattattcttaatcttaaaactataaatatggatcttttatgattctttttgtaagccctaggggctctaaaatggtttcacttATTTAtcccaaaatATTTAttattatgttgaatgttaaatatagtatctatgtagattggttagtaaaactATTTAataaatttgataaatataaa

The anti-CD19 RDE CAR and anti-CD19 CAR constructs are transfected byroutine methods into different populations of T-cells (primary humanT-cells), and stable populations of T-cells are selected usingappropriate antibiotics (or other selection schemes). T-cell populationswith anti-CD19 RDE CARs (CD19⁻/CD22⁻/CD3⁺) and T-cell populations withanti-CD19 CARs (CD19⁻/CD22⁻/CD3⁺) are activated by co-incubation withanti-CD3/CD28 beads and allowed to return to quiescent state afterdebeading.

Quiescent anti-CD19 RDE CAR T-cells are co-cultured withCD19⁺/CD22⁺/CD3⁻ Raji target cells at RDE CAR T-cell:Raji target ratiosof 2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The RDE-CAR T-cells and theRaji cells are grown together for 24 hours. Cultures are washed, andthen stained with anti-CD22 and anti-CD3 reagents, followed by countingof CD22⁺ (Raji target cells) and CD3⁺ cells (Smart CAR T-cells). Thesemeasurements will identify the target cell killing rate (e.g.,half-life) and the proliferation rate of the RDE-CAR T-cells atdifferent levels of RDE-CAR expression.

Activated anti-CD19 RDE CAR T-cells are co-cultured withCD19⁺/CD22⁺/CD3⁻ Raji target cells at RDE CAR T-cell:Raji target ratiosof 2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2, mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The RDE-CAR T-cells and theRaji cells are grown together for 24 hours. Samples from culture mediaare taken and tested for IL-2 by ELISA.

As a control activated anti-CD19 CAR T-cells are co-cultured withCD19⁺/CD22⁺/CD3⁻ Raji target cells at CAR T-cell:Raji target ratios of2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The CAR T-cells and the Rajicells are grown together for 24 hours. Cultures are washed, and thenstained with anti-CD22 and anti-CD3 reagents, followed by counting ofCD22⁺ (Raji target cells) and CD3⁺ cells (CAR T-cells).

As a control, activated anti-CD19 CAR T-cells are co-cultured withCD19⁺/CD22⁺/CD3⁻ Raji target cells at CAR T-cell:Raji target ratios of2:1, 5:1, and 10:1. The glycolysis activator glucose is added to theculture medium at concentrations in the range of 1.0 mM to 10 mM (1 mM,2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM and 10 mM). The CAR T-cells and the Rajicells are grown together for 48 hours. Samples from culture media aretaken and tested for IL-2 by ELISA.

Example 2: Removal of MicroRNA Binding Sites from an RDE

The AU-rich element from the 3′-UTR of IL-2 has mir-181 and mir 186microRNA binding sites. Different combinations of the microRNA siteswere removed from the 3′-UTR of IL-2. When the MIR186 micro-RNA siteswere removed from the 3′-UTR of IL-2 the dynamic range of expressionfrom constructs with this UTR increased 50 fold. The modified IL-2,3′-UTR replaces CTT in the sequence with GAA and is shown below (the newGAA is underlined in the sequence):

(SEQ ID NO: 22) taattaagtgatcccacttaaaacatatcaggccttctATTTATTTAaatATTTAaattttatATTTAttgttgaatgtatggtttgctacctattgtaactattattcttaatcttaaaactataaatatggatcttttatgattGAAtttgtaagccctaggggctctaaaatggtttcacttATTTAtcccaaaatATTTAttattatgttgaatgttaaatatagtatctatgtagattggttagtaaaactATTTAataaatttgataaatataaa

The AU-rich element from the 3′UTR of IFNg also has micro-RNA bindingsites characterized as mir-125. The sequence of the IFNg RDE is:

(SEQ ID NO: 23) tggttgtcctgcctgcaatatttgaattttaaatctaaatctATTTAttaatATTTAacattATTTAtatggggaatatatttttagactcatcaatcaaataagtATTTAtaatagcaacttttgtgtaatgaaaatgaatatctattaatatatgtattATTTAtaattcctatatcctgtgactgtctcacttaatcctttgttttctgactaattaggcaaggctatgtgattacaaggetttatctcaggggccaactaggcagccaacctaagcaagatcccatgggttgtgtgtttatttcacttgatgatacaatgaacacttataagtgaagtgatactatccagttactgccggtttgaaaatatgcctgcaatctgagccagtgetttaatggcatgtcagacagaacttgaatgtgtcaggtgaccctgatgaaaacatagcatctcaggagatttcatgcctggtgcttccaaatattgttgacaactgtgactgtacccaaatggaaagtaactcatttgttaaaattatcaatatctaatatatatgaataaagtgtaagttcacaacta

Different combinations of the micro-RNA sites were removed from the3′UTR of IFNg and tested for increased expression. When the mir125micro-RNA sites were removed from the 3′-UTR of IFN-γ the expressionrate from constructs with this UTR is increased.

Expression of GFP in T-cells, transfected with the RDE-GFP plus themicroRNA sites, is compared to expression of GFP in T-cells with theRDE-GFP in which the microRNA sites have been removed, followingactivation with CD3/CD28 beads for 24 hours. The removal of the microRNAsites increased expression of the GFP by a factor of between 2-5 after24 hours, relative to the cells with microRNA sites.

Example 3: Payload Delivery to DLBCL Using an Anti-CD19 CAR T-Cell

The anti-CD19 Smart CAR T-lymphocytes and anti-CD19 CAR T-celllymphocytes of Example 6 are used in this example. These CART-lymphocytes are further engineered to include a construct encoding aPD-1 inhibitor under the control of the 3′-UTR of IL2 that has beenmodified by removal of the MIR186 sites. PD-1 inhibitors expressed bythe construct include, for example, Pembrolizumab (Keytruda®), Nivolumab(Opdivo®), Atezolizumab (Tecentriq®), BMS-936558, Lambrolizumab, orpolypeptides derived from these drugs. Other PD-1 inhibitors that may beexpressed by the construct include those disclosed in Herbst et al., JClin Oncol., 31:3000 (2013); Heery et al., J Clin Oncol., 32:5s, 3064(2014); Powles et al., J Clin Oncol, 32:5s, 5011(2014); Segal et al., JClin Oncol., 32:5s, 3002 (2014), or

U.S. Pat. Nos. 8,735,553; 8,617,546; 8,008,449; 8,741,295; 8,552,154;8,354,509; 8,779,105; 7,563,869; 8,287,856; 8,927,697; 8,088,905;7,595,048; 8,168,179; 6,808,710; 7,943,743; 8,246,955; and 8,217,149.

T-cell populations with anti-CD19 Smart CARs/PD-1 (CD19−/CD22−/CD3+) andT-cell populations with anti-CD19 CARs/PD-1 (CD19−/CD22−/CD3+) areactivated by co-incubation with anti-CD3/CD28 beads. T-cells withanti-CD19 Smart CARs/PD-1 inhibitor or anti-CD19 CARs/PD-1 inhibitorwere incubated with theophylline at 0, 75 and 250 μM for 72 hours.Activated anti-CD19 Smart CAR/PD-1 T-cells or anti-CD19 CAR/PD-1 T-cellswere co-cultured with CD19+/CD22+/CD3− Raji target cells at SmartCAR/PD-1 T-cell:Raji target ratios of 2:1, 5:1, and 10:1. Ligand for theRNA control device, theophylline is maintained in the culture medium atconcentrations of 0 μM, 75 μM, and 250 μM. The Smart-CAR/PD-1 T-cells orCAR/PD-1 T-cells and the Raji cells are grown together for 18 hours.Cultures are washed, and then stained with anti-CD22 and anti-CD3reagents, followed by counting of CD22+ (Raji target cells) and CD3+cells (Smart CAR T-cells). Samples from culture media are also taken at6, 12 and 18 hours, and tested for PD-1 inhibitor by ELISA.

Example 4: Payload Delivery to AML Using an Anti-CD133 CAR T-Cell

A CAR is made using the anti-CD20 CAR cassette described in Budde 2013(Budde et al. PLoS1, 2013 doi:10.1371/journal.pone.0082742, which ishereby incorporated-by-reference in its entirety for all purposes), withthe anti-CD133 mAb 293C3-SDIE is used for the extracellular element(Rothfelder et al., 2015,ash.confex.com/ash/2015/webprogram/Paper81121.html, which isincorporated by reference in its entirety for all purposes) replacingthe anti-CD20 extracellular domain. The anti-CD133 CAR also can encodethe RNA control device, 3XL2bulge9 (Win and Smolke 2007 Proc. Natl Acad.Sci. 104 (36): 14283-88, which is hereby incorporated by reference inits entirety for all purposes). A nucleic acid encoding the anti-CD20CAR cassette is engineered to replace the anti-CD20 extracellular domainwith the anti-CD133 element, and optionally the RNA control device isalso engineered into the cassette. The anti-CD133 CAR with or withoutthe RNA control device are cloned into appropriate expression vectors.

These anti-CD133 CAR and anti-CD133 Smart CAR constructs are transfectedby routine methods into T-lymphocytes (Jurkat cells and/or primary humanT-lymphocytes), and stable populations of T-lymphocytes are selectedusing appropriate antibiotics (or other selection schemes).

These CAR T-lymphocytes are further engineered to include a constructencoding a PD-1 inhibitor under the control of the RDE from the 3′-UTRof IL2 that has been modified by removal of a MIR186 site. PD-1inhibitors expressed by the construct include, for example,Pembrolizumab (Keytruda®), Nivolumab (Opdivo®), Atezolizumab(Tecentriq®), BMS-936558, Lambrolizumab, or polypeptides derived fromthese drugs. Other PD-1 inhibitors that may be expressed by theconstruct include those disclosed in Herbst et al., J Clin Oncol.,31:3000 (2013); Heery et al., J Clin Oncol., 32:5s, 3064 (2014); Powleset al., J Clin Oncol, 32:5s, 5011(2014); Segal et al., J Clin Oncol.,32:5s, 3002 (2014), or U.S. Pat. Nos. 8,735,553; 8,617,546; 8,008,449;8,741,295; 8,552,154; 8,354,509; 8,779,105; 7,563,869; 8,287,856;8,927,697; 8,088,905; 7,595,048; 8,168,179; 6,808,710; 7,943,743;8,246,955; and 8,217,149.

T-lymphocyte populations with anti-CD133 CAR/PD-1 inhibitor oranti-CD133 Smart CAR/PD-1 inhibitor (CD20⁻/CD22⁻/CD3⁺) are activated byco-incubation with anti-CD3/CD28 beads.

Activated anti-CD133 CAR/PD-1 inhibitor or anti-CD133 Smart CAR/PD-1inhibitor T-lymphocytes are co-cultured with CD133⁺/CD3⁻ AML targetcells (e.g., U937, MV4-11, MOLM-14, HL-60 and/or KG1a) at anti-CD133 CARand/or anti-CD133 Smart CAR T-lymphocyte:AML target ratios of 2:1, 5:1,and 10:1. Ligand for the RNA control device, theophylline, is added tothe culture medium at concentrations in the range of 500 μM to 1 mM(lower or greater concentrations can be used to titrate Smart-CARactivity to the desired level). The anti-CD133 CAR/PD-1 inhibitor and/oranti-CD133 Smart CAR/PD-1 inhibitor T-lymphocytes and the AML cells aregrown together for 48 hours. Cultures are washed, and then stained withanti-CD133 and anti-CD3 reagents, followed by counting of CD133⁺ (AMLtarget cells) and CD3⁺ cells (anti-CD133 CAR, anti-CD133 DE-CAR,anti-CD133 Smart CAR, and/or the anti-CD133 DE-Smart CAR T-lymphocytes).These measurements will identify the target cell killing rate (e.g.,half-life) and the proliferation rate of the anti-CD133 CAR/PD-1inhibitor and/or anti-CD133 Smart CAR/PD-1 inhibitor T-lymphocytes atdifferent levels of CAR expression. Samples from culture media are alsotaken at 12, 24, 26 and 48 hours, and tested for PD-1 inhibitor byELISA.

Example 5: An RDE Construct for Expressing a Second Transgene

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a GFP-RDE1 (3′-UTR from IFNg) insert. These twoinserts/cassettes were placed in the same lenti virus construct. Theanti-CD19 CAR cassette and the insert with the GFP-RDE are transcribedin opposite directions, and the control regions for each are located inbetween the two insert/cassettes. The control region for the GFP-RDEinsert was MinP and the RDE was the endogenous 3′-UTR of IFNg. Thecontrol region of the anti-CD19 CAR cassette was MND (as describedabove). CD4+ T-cells were transduced with the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24 h alone in medium. For the‘Raji co-culture’ set and the “CD3/CD28 Beads” set, CD19+ Raji B cellsor anti-CD3/anti-CD28 beads were incubated with the transduced T cellsfor 24 h. At 24 h, the T cells were stained for CD25 and CD69, which areactivation markers, and subject to flow cytometry to measure thesemarkers and GFP expression in the T cells.

The transduced T-cells showed an increase in fluorescence when culturedwith Raji target cells (activate CAR) of 1.0% to 6.5% (about 6.5 fold),and increase in fluorescence when cultured with CD3/CD28 beads (activateTCR) of 1.0% to 4.4% (about 4.4 fold). The transformed T-cells showed achange in activated cells in the population when cultured with Rajicells of 0.9% to 84.8%, and when cultured with CD3/CD28 beads of 0.9% to90.8%.

Example 6: A Modified RDE2 Construct for Expressing a Second Transgene

Constructs were made using an anti-CD19 CAR cassette as described inExamples 11 and 12, and a GFP-RDE2.1 (IL-2 RDE) insert. The RDE2.1 wasmodified to remove the MIR186 microRNA sites, altering nucleotides fromthe 3′-UTR of IL-2 which was used as RDE2.

These two inserts/cassettes were placed in the same lenti virusconstruct. The anti-CD19 CAR cassette and the insert with the GFP-RDEare transcribed in opposite directions, and the control regions for eachare located in between the two insert/cassettes. The control region forthe GFP-RDE insert was a MinP. The control region of the anti-CD19 CARcassette in was MND (as described above). CD4+ T-cells were transducedwith the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24 h alone in medium. For the‘Raji co-culture’ set and the “CD3/CD28 Beads” set, CD19+ Raji B cellsor anti-CD3/anti-CD28 beads were incubated with the transduced T cellsfor 24 h. At 24 h, the T cells were stained for CD25 and CD69, which areactivation markers, and subject to flow cytometry to measure thesemarkers and GFP expression in the T cells.

The transduced T-cells showed a change in activated cells in thepopulation when cultured with Raji cells of 3.9% to 12.1%, and whencultured with CD3/CD28 beads of 3.9% to 11.1%.

Example 7: An RDE Construct for Expressing a Luciferase Transgene

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a Luciferase-RDE1 (3′-UTR of IFNg, Gold1) insertor a Luciferase-3′-UTR (a 3′-UTR that does not confer differentialtransgene translation in response to metabolic state of the cell,3′-UTR). The anti-CD19 CAR cassette and the insert with theluciferase-RDE1 are transcribed in opposite directions, and the controlregions for each are located in between the two insert/cassettes. Thecontrol region for the Luciferase-RDE1 insert and Luciferase-3′-UTR wereeither a MinP promoter or an NFAT promoter having the sequences of:

(MinP) SEQ ID NO: 24 TAGAGGGTATATAATGGAAGCTCGACTTCCAG (NFAT)SEQ ID NO: 25 GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTAGATCTAGACTCTAGAGGGTATATAATGGAAGCTCGAATTCThe control region of the anti-CD19 CAR cassette was the MND promoter.CD4+ T-cells were transduced with the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24 h alone in medium. For the‘Raji co-culture’ set and the “CD3/CD28 Beads” set, CD19+ Raji B cellsor anti-CD3/anti-CD28 beads were incubated with the transduced T cellsfor 24 h. At 24 h, the T cells were stained for CD25 and CD69, which areactivation markers, and subject to flow cytometry to measure thesemarkers and luciferase expression in the T cells.

FIG. 2 shows that the transduced T-cells had an increase inbioluminescence when cultured with Raji target cells (activate CAR) orwhen cultured with CD3/CD28 beads (activate TCR) as compared tobioluminescence of T-cells at resting. The T-cells with a NFAT promoterand the 3′-UTR of IFNg (Gold1) showed a larger on-off response from CARstimulation versus TCR stimulation. Under all conditions, T-cells withGold1 had lower amounts of bioluminescence than T-cells under the sameconditions (and same promoter) with Luciferase that was not controlledby the 3′UTR of IFNg (3′-UTR).

Example 8: Comparison of RDEs Controlling Luciferase

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a Luciferase-RDE1 (3′ UTR of IFNg, Gold1) insert,a Luciferase-RDE2 (3′-UTR of IL-2, Gold2) insert, a Luciferase-RDE3(3′-UTR of IL-2 modified as described above to remove the mir186 sites,Gold3), or a Luciferase-3′-UTR (a 3′-UTR that does not conferdifferential transgene translation in response to metabolic state of thecell, 3′-UTR). Combinations of these inserts/cassettes shown in FIG. 3were placed in the similar lenti virus constructs. The anti-CD19 CARcassette and the insert with the luciferase-RDE are transcribed inopposite directions, and the control regions for each are located inbetween the two insert/cassettes. The control region for theLuciferase-RDE insert and Luciferase-3′-UTR were either a MinP promoteror an NFAT promoter. The control region of the anti-CD19 CAR cassettewas the MND promoter, and CD4+ T-cells were transduced with thebicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24 h alone in medium. For the‘Raji co-culture’ set CD19+ Raji B cells were incubated with thetransduced T cells for 24 h. At 24 h, the T cells were stained for CD25and CD69, which are activation markers, and subject to flow cytometry tomeasure these markers and luciferase expression in the T cells.

FIG. 3 shows that the transduced T-cells had an increase inbioluminescence when cultured with Raji target cells (activate CAR) ascompared to bioluminescence of T-cells at resting for constructs withRDE1 (Gold1), RDE2 (Gold2), or RDE3 (Gold3). The T-cells with a NFATpromoter and the RDE1 showed a larger on-off response than T-cells witha MinP promoter and the corresponding RDE. Under all conditions, T-cellswith an RDE controlling luciferase had lower amounts of bioluminescencethan T-cells with luciferase cassettes that were not controlled by anRDE. Combined with the MinP promoter, RDE1 gave a 4.1-fold increase inbioluminescence with CAR stimulation, RDE2 gave a 1.8-fold increase inbioluminescence, and RDE3 gave a 1.4-fold increase. Combined with theNFAT promoter, RDE1 gave a 8.5-fold increase in bioluminescence with CARstimulation, RDE2 gave a 3.1-fold increase in bioluminescence, and RDE3gave a 1.3-fold increase. With either promoter, the RDE3 construct gavethe highest amount of bioluminescence, the RDE1 construct gave thelowest amount of bioluminescence, and the RDE2 construct gave an amountof bioluminescence between RDE3 and RDE1.

Example 9: An RDE Construct for Expressing IL-12

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and an IL-12-RDE1 (3′-UTR of IFNg) insert or an IL-123′-UTR (a 3′-UTR that does not confer differential transgene translationin response to metabolic state of the cell). The anti-CD19 CAR cassetteand the insert with the IL-12-RDE1 are transcribed in oppositedirections, and the control regions for each are located in between thetwo insert/cassettes. The control region for the IL-12-RDE1 insert andIL-12 3′-UTR were either a minP promoter or an NFAT promoter. Thecontrol region of the anti-CD19 CAR cassette was the MND promoter. CD4+T-cells were transduced with the bicistronic construct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24 h alone in medium. For the‘Raji co-culture’ set, CD19+ Raji B cells were incubated with thetransduced T cells for 24 h. At 24 h, the T cells were stained for CD25and CD69, which are activation markers, and subject to flow cytometry tomeasure these markers. IL-12 expression in the T cells was measured byELISA.

FIG. 4 shows that the transduced T-cells had an increase in IL-12expression when cultured with Raji target cells (activate CAR) ascompared to IL-12 expression of T-cells at resting using constructscontrolled by the MinP promoter or NFAT promoter. T-cells with the NFATpromoter and RDE1 (Gold1) showed a 168-fold change in IL-12 expressionform resting to CAR stimulation. T-cells with the NFAT promoter and a3′-UTR (not responsive to CAR stimulation, 3′-UTR) showed a 50-foldchange in expression, and a minP promoter with RDE1 (Gold1) showed a 6.3fold change in expression.

Example 10: AU Elements and Steady State Expression

Constructs were made with different RDEs operably linked to a nucleicacid encoding luciferase. The different RDEs used were AU 4 (CTLA4), AU13 (IL-5), AU 14 (IL-6), AU 15 (IL-9), AU 16 (IL-10), AU 17 (IL-13), andAU 101 (IFNg). These luciferase-AU constructs were transduced intoprimary T-cells. After the cells returned to the resting stage they wereplated and sham induced (basal) or induced with anti-CD3 and anti-CD28antibody (activated). At 24 hours post activation the amount ofluciferase units in each was measured. These amounts are plotted in thebar graph of FIG. 5.

The AU elements in this example had different basal expression levels,different induced expression levels (at 24 hours), and different levelsof fold induction. The AU constructs showed different amounts of basalexpression, different amounts of induced expression and differentamounts of fold induction (or dynamic range).

Example 11: AU Elements and Expression Parameters

Constructs were made with different RDEs operably linked to a nucleicacid encoding luciferase. The different RDEs used were AU 2 (CSF2), AU 3(CD247), AU 5 (EDN1), AU 7 (SLC2A1), AU 10 (Myc), AU 19 (TMEM-219), AU20 (TMEM-219snp), AU 21 (CCR7), AU 22 (SEM-A4D), AU 23 (CDC42-SE2), andAU 101 (IFNg). These luciferase-AU constructs were transduced intoprimary T-cells. After the cells returned to the resting stage they wereplated and either not treated (basal) or activated with anti-CD3 andanti-CD28 antibody (activated). At 24 hours post activation the amountof luciferase units in each was measured. These amounts are plotted inthe bar graph of FIG. 6. Alternatively, after the cells returned toresting stage they were plated into 96-well plates in quadruplicate formeasuring at each time point: 1 day, 3 days, 6 days and 8 days. Thecells were either not treated or activated with anti-CD3 and anti-CD28antibody, and luciferase activity was measured at 1 day, 3 days, 6 daysand 8 days. These results are plotted in the bar graph of FIG. 7, andshown in Table 1 below. FIG. 8 shows selected data plotted in a bargraph. The numbers in parentheses in Table 1 below are the LuciferaseUnits on Days 3, 6, and 8 divided by the Luciferase Units of Day 1.

TABLE 1 Luciferase Units AU Construct Day 1 Day 3 Day 6 Day 8 AU 2(CSF2) 60051 306035 (5) 578305 (10) 591953 (10) AU 101 (IFNg) 85816473395 (6) 724129 (8)  817447 (10) AU 5 (EDN1) 69391 613921 (9) 838040(12) 1023000 (15)  AU 3 (CD247) 44939  595753 (13) 961839 (21) 1116000(25)  AU 20 (TMEM-219snp) 1135000 10750000 (9)  21020000 (19)  25480000(22)  AU 10 (Myc) 1233000 16020000 (13)  26780000 (22)  27800000 (23) AU 7 (SLC2A1) 4914  80906 (16) 132974 (27) 136537 (28) AU 21 (CCR7)27128  465140 (17) 604016 (22) 692715 (26) AU 23 (CDC42-SE2) 711051215000 (17) 2012000 (28)  2110000 (30)  AU 22 (SEM-A4D) 226815 2829000(12) 6106000 (27)  7396000 (33)  AU 19 (TMEM-219) 833146 11260000 (14) 22560000 (27)  27500000 (33) 

The AU elements in FIG. 6, FIG. 7 and Table 1 had different basalexpression levels, different induced expression levels (at 24 hours),and different levels of fold induction. The basal expression levelsdiffered over an about 2000 fold range for these AU elements (AU 7 to AU20), and the induced expression levels differed over an about 5500 foldrange (AU 7 to AU 10). Basal expression for the constructs ranged from1390 for AU 7 (SLC2A1) to 2,927,000 for AU 20 (TMEM-219snp). Activatedexpression ranged from 4914 for AU 7 (day 1) to 27,800,0000 for AU 10(day 8). FIG. 8 and Table 1 show that some AU elements had lower levelsof output, for example, AU 101 (IFNg), AU 2 (CSF2), AU 5 (EDN1), AU 7(SLC2A1), AU 21 (CCR7), and AU 23 (CDC42-SE2). Some AU elements hadintermediate amounts of output: AU 19 (TMEM-219) and AU 22 (SEM-A4D).And some AU element had high output: AU 20 (TMEM-219snp) and AU 10(Myc).

The Luciferase data was also analyzed for dynamic range (fold inductionor luciferase activated/luciferase basal) of each luciferase-AUconstruct. The dynamic range (fold induction) for each AU construct atDays 1, ¾ (activated expression was measured on Day 3 and basalexpression was measured on Day 4), 6 and 8. This data is shown below inTable 2, and plotted in bar graphs in FIG. 9 and FIG. 10.

TABLE 2 Fold Induction AU Construct Day 1 Day 3/4* Day 6 Day 8 AU 2 9.39.3 8.1 5.8 AU 101 16.9 14.6 12.3 10.5 AU 5 5.7 11.9 10.1 9.4 AU 21 5.627.3 28.6 29.6 AU 3 1.8 6.7 8.1 6.8 AU 20 3.1 7.3 9.3 8.7 AU 10 3.7 13.214.1 10.2 AU 7 3.5 12.3 13.1 10.6 AU 23 2.6 12.8 15.1 12.2 AU 19 2.3 9.312.9 13.8 AU 22 1.2 4.6 6.9 7.1 *Induction was measured on Day 3 andbasal was measured on Day 4.

At Day 1 dynamic range (fold induction=activated/basal) ranged fromabout 1 (AU 22) to about 17 (AU 101). At Day ¾, dynamic range variedfrom about 4.5 (AU 22) to about 27 (AU21). At Day 6, dynamic rangevaries from about 7 (AU 22) to about 29 (AU 21). On Day 8, dynamic rangevaried from about 7 (AU22) to about 30 (AU 21). The AU constructs showeda number of related patterns. AU 2 and AU 101 showed a rapid increase indynamic range on Day 1, and then the dynamic range decreased on days 6and 8. AU 5 and AU 21 show increasing dynamic range from day 0 to day ¾,and then the dynamic range is maintained through days 6 and 8. AU 3, AU20, AU 10, AU 7 and AU 23 showed rising dynamic range from day 0 to day6, and then the dynamic range decreased on day 8. AU 19, and AU 22,showed rising dynamic ranges from day 0 to day 8.

AU 21 and AU 23 showed accelerating dynamic range and these AUconstructs also had low basal expression (day 1=4865 and 27363,respectively). AU 2 and AU 101 showed decreasing dynamic range from 24hours to 72 hours and these AU elements also had low basal expression.AU 5 and AU 20 also showed decreasing dynamic range from day 1 to day ¾(though more expression than AU 2 and AU 101) and AU 5 had low basalexpression whereas AU 20 had high basal expression. AU 10, AU 19 and AU22 showed consistent dynamic range from day 1 to day ¾ and had highbasal levels of expression. AU 3 and AU 7 also had consistent dynamicrange from day 1 to day ¾ and had low basal expression levels.

The above data shows that different AU elements have different temporaleffects on expression from days 1-8. Some AU elements show acceleratingdynamic range over different portions of the time range. The AU elementsshow different amounts of total expression (C_(max)) and different timesto maximum expression (T_(max)). The AU elements also show differentmaximum dynamic ranges and time to reach these maximums. These differingkinetics of expression can be used to provide customized basal, C_(max),T_(max), dynamic range, and time to max dynamic range for a desiredtransgene. These differing kinetics can also be used to providetemporally distinct expression for two transgenes in a cell afteractivation of the cell.

Example 12: AU Element Control with Glucose and Galactose

Constructs were made with different RDEs operably linked to a nucleicacid encoding luciferase. The RDE was an AU element responsive toglycolytic state of the cell. The AU element—luciferase constructs weretransduced into T-cells. After the cells reached the resting state, theywere split into wells and fed media including either glucose orgalactose. Luciferase activity was measured on days 3 and 5. Theseresults are shown in the bar graph of FIG. 11. The results show thatglucose increased expression of luciferase compared to galactose and theamount of expression increased from days 3 to 5. On day 3 the glucosetreated cells had 15× more expression of luciferase than the galactosetreated cells and on day 5 this had grown to 27× more expression.

Example 13: Delivery and Design of a Viral Payload

A Smart Car is made using the third generation anti-CD19 CAR cassettedescribed in WO 2012/079000, which is hereby incorporated-by-referencein its entirety for all purposes), and the RNA control device,3XL2bulge9 (Win and Smolke 2007 Proc. Natl Acad. Sci. 104 (36):14283-88, which is hereby incorporated by reference in its entirety forall purposes). A nucleic acid encoding the 3XL2bulge9 control device isengineered into the anti-CD19 CAR cassette in an appropriate expressionvector. The anti-CD19 Smart CAR and anti-CD19 CAR constructs aretransfected by routine methods into different populations of T-cells(Jurkat cells and/or primary human T-cells), and stable populations ofT-cells are selected using appropriate antibiotics (or other selectionschemes). T-cell populations with anti-CD19 Smart CARs(CD19⁻/CD22⁻/CD3⁺) and T-cell populations with anti-CD19 CARs(CD19⁻/CD22⁻/CD3⁺) are activated by co-incubation with anti-CD3/CD28beads.

Third generation Lentiviral packaging, envelope, and transfer plasmidsare obtained from addgene. The Rev encoding packaging plasmid isengineered to include the AU101 (INFg) RDE in the 3′-UTR of Rev. Themodified Rev packaging plasmid, the Gag Pol packaging plasmid, and theenvelope plasmid are transfected into anti-CD19 T-lymphocyte cells. Atransfer plasmid is engineered to include GFP as the transgene in thetransfer plasmid. This transfer plasmid is also transfected into theanti-CD19 CAR T-lymphocyte cells.

Anti-CD19 Smart CAR T-lymphocytes are co-cultured with CD19+/CD22+/CD3−Ramos target cells at Smart CAR T-lymphocyte:Raji target ratios of 2:1,5:1, and 10:1. Ligand for the RNA control device, theophylline is addedto the culture medium at concentrations in the range of 2 μM to 2 mM (2μM, 10 μM, 20 μM, 100 μM, 200 μM, 1 mM, and 2 mM). The Smart-CAR T-cellsand the Raji cells are grown together for 48 hours.

At the end of the incubation period, the culture media is separated fromthe T-lymphocytes and Raji cells. Viral titer in the supernatant ismeasured using an ELISA with anti-lentivirus antibody reagents.Infectivity and payload delivery by the viruses is tested by infectingHEK 293 cells with the virus, and after a suitable incubation timemeasuring GFP fluorescence from the transduced HEK 293 cells.

Example 14: Payload Delivery Using Gold in a Mouse Lymphoma Model

Constructs were made using an anti-CD19 CAR cassette as described in WO2012/079000, which is hereby incorporated-by-reference in its entiretyfor all purposes), and a Luciferase-AU (3′ UTR of IL-6) insert. Theseconstructs were placed in a bicistronic lenti virus construct. Theanti-CD19 CAR cassette and the insert with the luciferase-RDE aretranscribed in opposite directions on the bicistronic vector, and thecontrol regions for each are located in between the twoinsert/cassettes. The control region for the Luciferase-RDE insert was aMinP promoter. The control region of the anti-CD19 CAR cassette was theMND promoter. CD4+ T-cells were transduced with the bicistronicconstruct.

The transduced T cells were allowed to return to resting state, and thenwere tested after stimulation as follows. For the ‘no stimulation’ set,transduced T-cells were incubated for 24 h alone in medium. For the‘Raji co-culture’ set CD19+ Raji B cells were incubated with thetransduced T cells for 24 h. At 24 h, the T cells were stained for CD25and CD69, which are activation markers, and subject to flow cytometry tomeasure these markers and luciferase expression in the T cells. These invitro results showed that the anti-CD19 CAR T-cells made luciferaseafter activation of the T-cells through the CAR.

These anti-CD19 CAR T-cells were also tested in a mouse model forlymphoma. CD19+ Raji cells were implanted in the flanks of NSG mice.After tumor formation, the anti-CD19 CAR T-cells were injected into themice and the mice were scanned for luminescence. Imaging of the miceshowed luminescence at the tumor sites from anti-CD19 CAR T-cells thathave been activated by the CD19 positive tumor. The amount ofluminescence increased over time as more T-cells were activated.

Example 15: Payload Delivery to αvβ6 Positive Solid Tumor

A nucleic acid encoding a knottin as described in Silverman et al., J.Mol. Biol. 385:1064-75 (2009) and Kimura et al, Proteins 77:359-69(2009), which are incorporated by reference in their entirety for allpurposes is operably linked to a nucleic acid encoding the CARcomponents aCD43z,CD8Hinge,CD8transmembrane,41BB(CD28 or other costim),and CD3z to make a nucleic acid encoding an anti-αvβ6 CAR.

The nucleic acid encoding the anti-αvβ6 CAR is transfected by routinemethods into T-cells (Jurkat cells and/or primary human T-cells), andstable populations of T-cells are selected using appropriate antibiotics(or other selection schemes). T-cell populations with anti-αvβ6 CARs areactivated by co-incubation with anti-CD3/CD28 beads. These cells arealso engineered with an expression cassette encoding IL-12 operablylinked to the Gold element from INFg or AU 21 (CCR7) is placed under thecontrol of the promoter Min P.

The anti-αvβ6 CAR T-cells are incubated in wells with αvβ6 tumor cells.After incubation, the wells are tested for secretion of IL-12 from theanti-αvβ6 CAR T-cells. anti-αvβ6 CAR T-cells secrete IL-12 whenincubated with αvβ6 tumor cells, and the controls show low or nosecretion when the CAR T-cell is not stimulated.

Example 16: An Anti-Onco CD 43 CAR for AML

A single chain antibody for onco-sialylated CD 43 was made using ananti-onco-sialylated CD 43 antibody. The nucleic acid encoding thissingle-chain antibody was combined with a nucleic acid encoding the CARcomponents aCD43z,CD8Hinge,CD8transmembrane,41BB(CD28 or other costim),and CD3z to make a nucleic acid encoding an anti-onco-sialylated CD 43CAR.

The nucleic acid encoding the anti-onco-sialylated CD 43 CAR istransfected by routine methods into T-cells (Jurkat cells and/or primaryhuman T-cells), and stable populations of T-cells are selected usingappropriate antibiotics (or other selection schemes). T-cell populationswith anti-onco-sialylated CD 43 CARs are activated by co-incubation withanti-CD3/CD28 beads.

Example 17: Payload Delivery to CD 43 Positive AML

An expression cassette encoding IL-12 operably linked to the Goldelement from INFg or AU 21 (CCR7) is placed under the control of thepromoter Min P, and engineered into the anti-onco-sialylated CD 43 CART-cell.

The anti-onco-sialylated CD 43 CAR T-cells are incubated in wells withAML cells. After incubation, the wells are tested for secretion of IL-12from the anti-onco-sialylated CD 43 CAR T-cells. Anti-onco-sialylated CD43 CAR T-cells secrete IL-12 when incubated with AML cells, and thecontrols show low or no secretion when the CAR T-cell is not stimulated.

All publications, patents and patent applications discussed and citedherein are incorporated herein by reference in their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method of controlling expression of atransgene, comprising the steps of: obtaining a primary T-cellcomprising a chimeric antigen receptor, and a heterologous nucleic acidcomprising a polynucleotide encoding the transgene that is operablylinked to a polynucleotide encoding a RNA degradation element (RDE),wherein the RDE is an AU101 (Interferon gamma or IFNg) or an AU14 (IL6),wherein the heterologous nucleic acid is transcribed to make atranscript encoding the transgene operably linked to the RDE, wherein anRDE binding protein binds to the RDE and regulates expression of thetransgene; exposing the primary T-cell to a ligand for the chimericantigen receptor wherein binding of the ligand by the chimeric antigenreceptor activates the primary T-cell and the primary T-cellproliferates, and the activation of the primary T-cell changes bindingof the RDE by the RDE binding protein; and expressing the transgenewherein the transgene has increased levels of expression after theactivation of the primary T-cell compared to the level of expression ofthe transgene before activation of the primary T-cell.
 2. The method ofclaim 1 wherein the transgene encodes a cytokine, a FasL, an antibody, agrowth factor, a chemokine, an enzyme that cleaves a polypeptide or apolysaccharide, a granzyme, a perforin, or a checkpoint inhibitor. 3.The method of claim 1, wherein the transgene encodes an IL-2, and IL-12,an IL-15, an IL-18 or a TNF-α.
 4. The method of claim 3, wherein thetransgene encodes an IL-2.
 5. The method of claim 3, wherein thetransgene encodes an IL-12.
 6. The method of claim 3, wherein thetransgene encodes an IL-15.
 7. The method of claim 3, wherein thetransgene encodes an IL-18.
 8. The method of claim 3, wherein thetransgene encodes a TNF-α.
 9. The method of claim 2, wherein thetransgene encodes an anti-4-1BB antibody.
 10. The method of claim 1,wherein the transgene encodes a CD40L.
 11. The method of claim 1,wherein the transgene encodes a Hsp60 or a Hsp70.
 12. The method ofclaim 11, wherein the transgene encodes a Hsp60.
 13. The method of claim11, wherein the transgene encodes a Hsp70.
 14. The method of claim 1,wherein the chimeric antigen receptor is an anti-DLL3 chimeric antigenreceptor.
 15. The method of claim 14, wherein the ligand is a DLL3 foundon a target cell.
 16. The method of claim 15, wherein the target cell isa tumor cell.
 17. The method of claim 16, wherein the tumor cell is asmall cell lung cancer cell.
 18. The method of claim 16, wherein thetumor cell is a melanoma cell.
 19. The method of claim 16, wherein thetumor cell is an IDH1 mutant glioma cell.